ErbB4 receptor-specific neuregulin related ligands and uses therefor

ABSTRACT

The invention concerns a novel neuregulin related ligand (NRG3) including fragments and variants thereof, as new members of the neuregulin family of compounds. The invention also concerns methods and means for producing NRG3. The native polypeptides of the invention are characterized by containing an extracellular domain including an EGF-like domain, a transmembrane domain and a cytoplasmic domain. Isolated nucleotide sequences encoding such polypeptides, expression vectors containing the nucleotide sequences, recombinant host cells transformed with the vectors, and methods for the recombinant production for the novel NRG3s are also within the scope of the invention.

RELATED APPLICATION

[0001] This application is a non-provisional application filed under 37CFR 1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/052,019 filed Jul. 9, 1997, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention concerns novel neuregulin related ligands.More particularly, the invention relates to a new member of theneuregulin family and functional derivatives of the novel polypeptide.

BACKGROUND OF THE INVENTION

[0003] Signal transduction affecting cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates. Growth factor receptor protein tyrosine kinases of the classI subfamily include the 170 kDa epidermal growth factor receptor (EGFR)encoded by the erbB 1 gene. erbB 1 has been causally implicated in humanmalignancy. In particular, increased expression of this gene has beenobserved in more aggressive carcinomas of the breast, bladder, lung andstomach. The second member of the class I subfamily, p185^(neu), wasoriginally identified as the product of the transforming gene fromneuroblastomas of chemically treated rats. The neu gene (also callederbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase.Amplification and/or overexpression of the human HER2 gene correlateswith a poor prognosis in breast and ovarian cancers (Slamon et al.,(1987) Science 235:177-182; and Slamon et al., (1989) Science244:707-712). Overexpression of HER2 has been correlated with othercarcinomas including carcinomas of the stomach, endometrium, salivarygland, lung, kidney, colon and bladder. A further related gene, callederbB3 or HER3, has also been described (Kraus et al., (1989) Proc. Natl.Acad. Sci. USA 86:9193-9197). Kraus et al. (1989) discovered thatmarkedly elevated levels of erbB3 mRNA were present in certain humanmammary tumor cell lines indicating that erbB3, like erbB1 and erbB2,may play a role in human malignancies. The erbB3 gene has been found tobe overexpressed in breast (Lemoine et al. (1992) Br. J. Cancer66:1116-1121), gastrointestinal (Poller et al. (1992) J. Pathol.168:275-280, Rajkumer et al. (1993) J. Pathol. 170:271-278, and Sanidaset al. (1993) Int. J. Cancer 54:935-940, and pancreatic cancers (Lemoineet al. (1992) J. Pathol. 168:269-273, and Friess et al. (1995) ClinicalCancer Research 1:1413-1420).

[0004] The class I subfamily of growth factor receptor protein tyrosinekinases has been further extended to include the HER4/Erb4 receptor (EPPat Appln No 599,274; Plowman et al. (1993) Proc. Natl. Acad. Sci. USA90:1746-1750; and Plowman et al. (1993) Nature 366:473-475. Plowman etal. found that increased HER4 expression closely correlated with certaincarcinomas of epithelial origin, including breast adenocarcinomas.Diagnostic methods for detection of human neoplastic conditions(especially breast cancers) which evaluate HER4 expression are describedin EP Pat Appln No. 599,274.

[0005] The quest for the activator of the HER2 oncogene has lead to thediscovery of a family of polypeptides, collectively called neuregulins(NRG1). These proteins appear to result from alternate splicing of asingle gene which was mapped to the short arm of human chromosome 8 byOrr-Urtreger et al. (1993) Proc. Natl. Acad. Sci. USA 90:1867-1871.

[0006] Holmes et al. isolated and cloned a family of polypeptideactivators for the HER2 receptor which they called heregulin-α (HRG-α),heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like(HRG-β2-like), and heregulin-β3 (HRG-β3). See Holmes et al.(1992)Science 256:1205-1210; WO 92/20798; and U.S. Pat. No. 5,367,060. The 45kDa polypeptide, HRG-α, was purified from the conditioned medium of theMDA-MB-231 human breast cancer cell line. These researchers demonstratedthe ability of the purified heregulin polypeptides to activate tyrosinephosphorylation of the HER2 receptor in MCF7 breast tumor cells.Furthermore, the mitogenic activity of the heregulin polypeptides onSK-BR-3 cells (which express high levels of the HER2 receptor) wasillustrated. Like other growth factors which belong to the EGF family,soluble HRG polypeptides appear to be derived from a membrane boundprecursor (called pro-HRG) which is proteolytically processed to releasethe 45 kDa soluble form. These pro-HRGs lack a N-terminal signalpeptide.

[0007] While heregulins are substantially identical in the first 213amino acid residues, they are classified into two major types, α and β,based on two variant EGF-like domains which differ in their C-terminalportions. Nevertheless, these EGF-like domains are identical in thespacing of six cysteine residues contained therein. Based on an aminoacid sequence comparison, Holmes et al. found that between the first andsixth cysteines in the EGF-like domain, HRGs were 45% similar toheparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

[0008] The 44 kDa neu differentiation factor (NDF), which is the ratequivalent of human HRG, was first described by Peles et al. (1992) Cell69:205-216; and Wen et al. (1992) Cell 69:559-572. Like the HRGpolypeptides, NDF has an immunoglobulin (Ig) homology domain followed byan EGF-like domain and lacks a N-terminal signal peptide. Subsequently,Wen et al. (1994) Mol. Cell. Biol. A4(3):1909-1919 carried out cloningexperiments to extend the family of NDFs. This work revealed sixdistinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes etal., the NDFs are classified as either α or β, polypeptides based on thesequences of the EGF-like domains. Isoforms 1 to 4 are characterized onthe basis of the region between the EGF-like domain and transmembranedomain. Also, isoforms a, b and c are described which have variablelength cytoplasmic domains. These researchers conclude that differentNDF isoforms are generated by alternative splicing and perform distincttissue-specific functions. See also EP 505 148; WO 93/22424; and WO94/28133 concerning NDF.

[0009] Falls et al. (1993) Cell 72:801-815 describe another member ofthe heregulin family which they call acetylcholine receptor inducingactivity (ARIA) polypeptide. The chicken-derived ARIA polypeptidestimulates synthesis of muscle acetylcholine receptors. See also WO94/08007. ARIA is a β-type heregulin and lacks the entire spacer regionrich in glycosylation sites between the Ig-like domain and EGF-likedomain of HRGβ, and HRGβ1-β3.

[0010] Marchionni et al. identified several bovine-derived proteinswhich they call glial growth factors (GGFs) (Marchionni et al. (1993)Nature 362:312-318). These GGFs share the Ig-like domain and EGF-likedomain with the other heregulin proteins described above, but also havean amino-terminal kringle domain. GGFs generally do not have thecomplete glycosylated spacer region between the Ig-like domain andEGF-like domain. Only one of the GGFs, GGFII, possessed a N-terminalsignal peptide. See also WO 92/18627; WO 94/00140; WO 94/04560; WO94/26298; and WO 95/32724 which refer to GGFs and uses thereof.

[0011] Ho et al. in (1995) J. Biol. Chem. 270(4):14523-14532 describeanother member of the heregulin family called sensory and motorneuron-derived factor (SMDF). This protein has an EGF-like domaincharacteristic of all other heregulin polypeptides but a distinctN-terminal domain. The major structural difference between SMDF and theother heregulin polypeptides is the lack in SMDF of the Ig-like domainand the “glyco” spacer characteristic of all the other heregulinpolypeptides. Another feature of SMDF is the presence of two stretchesof hydrophobic amino acids near the N-terminus.

[0012] While the heregulin polypeptides were first identified based ontheir ability to activate the HER2 receptor (see Holmes et al., supra),it was discovered that certain ovarian cells expressing neu andneu-transfected fibroblasts did not bind or crosslink to NDF, nor didthey respond to NDF to undergo tyrosine phosphorylation (Peles et al.(1993) EMBO J. 12:961-971). This indicated that another cellularcomponent was necessary for conferring full heregulin responsiveness.Carraway et al. subsequently demonstrated that ¹²⁵I-rHRGβ1₁₇₇₋₂₄₄ boundto NIH-3T3 fibroblasts stably transfected with bovine erbB3 but not tonon-transfected parental cells. Accordingly, they conclude that ErbB3 isa receptor for HRG and mediates phosphorylation of intrinsic tyrosineresidues as well as phosphorylation of ErbB2 receptor in cells whichexpress both receptors. Caraway et al. (1994) J. Biol. Chem.269(19):14303-14306. Sliwkowski et al. (1994) J. Biol. Chem.269(20):14661-14665 found that cells transfected with HER3 alone showlow affinities for heregulin, whereas cells transfected with both HER2and HER3 show higher affinities.

[0013] This observation correlates with the “receptor cross-talking”described previously by Kokai et al., Cell 58:287-292 (1989); Stem etal. (1988) EMBO J. 7:995-1001; and King et al., 4:13-18 (1989). Theseresearchers found that binding of EGF to the EGFR resulted in activationof the EGFR kinase domain and cross-phosphorylation of p185^(HER2). Thisis believed to be a result of ligand-induced receptor heterodimerizationand the concomitant cross-phosphorylation of the receptors within theheterodimer (Wada et al. (1990) Cell 61:1339-1347).

[0014] Plowman and his colleagues have similarly studiedp185^(HER4)/p185^(HER2) activation. They expressed p185^(HER2) alone,p185^(HER4) alone, or the two receptors together in human T lymphocytesand demonstrated that heregulin is capable of stimulating tyrosinephosphorylation of p185^(HER4), but could only stimulate p185^(HER2)phosphorylation in cells expressing both receptors. Plowman et al.,Nature 336:473-475 (1993). Thus, heregulin is the only known example ofa member of the EGF growth factor family that can interact with severalreceptors. Carraway and Cantley (1994) Cell 78:5-8.

[0015] The biological role of heregulin has been investigated by severalgroups. For example, Falls et al., (discussed above) found that ARIAplays a role in myotube differentiation, namely affecting the synthesisand concentration of neurotransmitter receptors in the postsynapticmuscle cells of motor neurons. Corfas and Fischbach demonstrated thatARIA also increases the number of sodium channels in chick muscle.Corfas and Fischbach (1993) J. Neuroscience 13(5): 2118-2125. It hasalso been shown that GGFII is mitogenic for subconfluent quiescent humanmyoblasts and that differentiation of clonal human myoblasts in thecontinuous presence of GGFII results in greater numbers of myotubesafter six days of differentiation (Sklar et al. (1994) J. Cell Biochem.,Abst. W462, 18D, 540). See also WO 94/26298 published Nov. 24, 1994.

[0016] Holmes et al., supra, found that HRG exerted a mitogenic effecton mammary cell lines (such as SK-BR-3 and MCF-7). The mitogenicactivity of GGFs on Schwann cells has also been reported. See, e.g.,Brockes et al. (1980) J. Biol. Chem. 255(18):8374-8377; Lemke andBrockes (1984) J. Neurosci. 4:75-83; Brockes et al. (1984) J.Neuroscience 4(1):75-83; Brockes et al. (1986) Ann. Neurol.20(3):317-322; Brockes, J. (1987) Methods in Enzym. 147:217-225 andMarchionni et al., supra. Schwann cells constitute important glial cellswhich provide myelin sheathing around the axons of neurons, therebyforming individual nerve fibers. Thus, it is apparent that Schwann cellsplay an important role in the development, function and regeneration ofperipheral nerves. The implications of this from a therapeuticstandpoint have been addressed by Levi et al. (1994) J. Neuroscience14(3):1309-1319. Levi et al. discuss the potential for construction of acellular prosthesis comprising human Schwann cells which could betransplanted into areas of damaged spinal cord. Methods for culturingSchwann cells ex vivo have been described. See WO 94/00140 and Li et al.(1996) J. Neuroscience 16(6):2012-2019.

[0017] Pinkas-Kramarski et al. found that NDF seems to be expressed inneurons and glial cells in embryonic and adult rat brain and primarycultures of rat brain cells, and suggested that it may act as a survivaland maturation factor for astrocytes (Pinkas-Kramarski et al. (1994)PNAS, USA 91:9387-9391). Meyer and Birchmeier (1994) PNAS, USA91:1064-1068 analyzed expression of heregulin during mouse embryogenesisand in the perinatal animal using in situ hybridization and RNaseprotection experiments. These authors conclude that, based on expressionof this molecule, heregulin plays a role in vivo as a mesenchymal andneuronal factor. Also, their findings imply that heregulin functions inthe development of epithelia. Similarly, Danilenko et al. (1994)Abstract 3101, FASEB 8(4-5 :A535, found that the interaction of NDF andthe HER2 receptor is important in directing epidermal migration anddifferentiation during wound repair.

[0018] Although NRG1 was initially proposed to be the ligand for thereceptor tyrosine kinase ErbB2, further studies have demonstrated thatactivation of ErbB2 frequently occurred as a result of NRG1 binding toErbB3 (Sliwkowski, M. X., et al. (1994) J. Biol. Chem. 269:14661-14665)or ErbB4 (Plowman, G. D. et al. (1993) Nature 366:473-475; and Carraway,K. L. and Cantley, L.C. (1994) Cell 78:5-8) receptors. Recent studieshave begun to highlight the roles of NRG1, ErbB2 receptor and ErbB4receptor in the development of the heart. Mice lacking ErbB4 receptor,ErbB2 receptor or NRG1 die during mid-embryogenesis (embryonic day 10.5)from the aborted development of myocardial trabeculae in the ventricle(Meyer & Birchmeier (1995) Nature 378:386-90; Gassmann et al. (1995)Nature 378:390-4; and Lee et al. (1995) Nature 378:394-8). These resultsare consistent with the view that NRG 1, expressed in the endocardium,is an important ligand required for the activation of ErbB2 and ErbB4receptors in the myocardium.

[0019] These same studies suggest that NRG1 and ErbB2 receptor may playa different role than ErbB4 receptor in the development of the hindbrain. NRG1 is expressed in the neuroepithelium and cells arising fromrhombomeres 2, 4 and 6, while ErbB4 receptor is expressed in rhombomeres3 and 5. NRG1 and ErbB2 receptor knockout mice exhibit a loss of cellsand axons of the cranial sensory ganglia. In contrast, ErbB4 receptordeficient mice do not exhibit a loss of cellularity in the cranialganglia. Rather, the organization, spacing and pattern of innervation ofthese ganglia to and from the central nervous system is disrupted(Gassmann et al., supra). One possible reason for this difference inhindbrain phenotypes of NRG1 and ErbB4 receptor knockout mice is thatadditional ligand(s) distinct from NRG1 may be recognized by ErbB4 inthe CNS (Gassmann et al., supra).

SUMMARY OF THE INVENTION

[0020] The present invention is based on the identification, recombinantproduction and characterization of a novel member of the family ofneuregulins (NRG1). More specifically, the invention concerns a novelpolypeptide, NRG3, comprising an EGF-like domain distinct from EGF-likedomains of NRG1 and NRG2. In addition, the NRG3 disclosed hereindisplays distinct receptor binding characteristics relative to otherneuregulin-like polypeptides.

[0021] In analyzing the homologous sequence motif, homology to theEGF-like domain of NRG1 was observed in the subset of amino acids thatare conserved in most neuregulins. Based upon this observation and theobserved ErbB4 receptor binding characteristics, the novel protein,NRG3, has been identified as a new member of the family of neuregulins.The novel protein contains domains that are distantly related to, butdistinct from, those found in the other members of the NRG1 family. Inaddition, it is expressed primarily in embryonic and adult tissues ofthe central nervous system. NRG3 represents a novel member of theneuregulin family of. compounds, members of which are involved in cellproliferation and differentiation, epithelial development, cardiacdevelopment, neurological development, as well as acting as glial cellmitogens, and as mesenchymal and neuronal factors.

[0022] In one aspect, the present invention concerns a novel isolatedmammalian NRG3 polypeptide having an EGF-like domain, and functionalderivatives of the novel NRG3, which polypeptides bind the ErbB4receptor. The native polypeptides within the scope of the presentinvention are characterized as containing an extracellular domainincluding an EGF-like domain, a transmembrane domain and a cytoplasmicdomain. The present invention specifically includes the soluble forms ofthe novel NRG3 ligand molecules of the invention, which have atransmembrane domain that cannot associate with a cell membrane, andoptionally devoid of all or part of the cytoplasmic domain. By“transmembrane domain” is meant a domain of the polypeptide thatcontains a sufficient number of hydrophobic amino acids to allow thepolypeptide to insert and anchor in a cell membrane. By “transmembranedomain that cannot associate with a cell membrane” is meant atransmembrane domain that has been altered by mutation or deletion suchthat is insufficiently hydrophobic to allow insertion or otherassociation with a cell membrane. Such a transmembrane domain does notpreclude, for example, the fusion of the NRG3 of the invention, orfragment thereof, with a secretion signal sequence useful for secretionof the polypeptide from the cell, an insufficient number of hydrophobicamino acid side chains are present devoid of an active transmembranedomain does not insert into a cell membrane. Mutations or alterations ofthe amino acid sequence useful to achieve an inactive transmembranedomain include, but are not limited to, deletion or substitution ofamino acids within the transmembrane domain. In a particular embodiment,the invention concerns isolated polypeptides, preferably NRG3 ligands,having at least 75% amino acid identity to polypeptides selected fromthe group consisting of

[0023] (1) a polypeptide comprising the amino acid sequence encoding theEGF-like domain shown in FIG. 3 (SEQ ID NO: 4);

[0024] (2) a polypeptide comprising the amino acid sequence encoding theextracellular domain of mouse or human NRG3 shown in FIG. 3 (SEQ ID NO:3 or SEQ ID NO: 7, respectively);

[0025] (3) a polypeptide comprising the amino acid sequence of thenative mouse or human NRG3 polypeptide shown in FIG. 3 (SEQ ID NO: 2 andSEQ ID NO: 6, respectfully);

[0026] (4) a further mammalian homologue of polypeptide (1)-(3);

[0027] (5) a soluble form of any of the polypeptides (1)-(4) devoid ofan active transmembrane domain; and

[0028] (6) a derivative of any of the polypeptides (1)-(5), retainingthe qualitative EGF-like domain and NRG3 receptor binding properties ofa polypeptide (1)-(5).

[0029] While the native NRG3 polypeptides of the present invention areglycoproteins, the present invention also encompasses variant moleculesunaccompanied by native glycosylation or having a variant glycosylationpattern. Preferably, the EGF-like domain of the NRG3 polypeptide isunglycosylated.

[0030] In a further embodiment, the invention includes an antagonist ofa novel NRG3 of the present invention. The antagonist of the inventionmay be a peptide that binds an NRG3 such as an anti-NRG3 antibody orbinding fragment thereof. Preferably, the NRG3 antagonist of theinvention substantially reduces binding of a natural ErbB4 receptorligand, such as an NRG3, to the ErbB4 receptor, thereby preventing orlimiting activation of the receptor. In a preferred embodiment, theantagonist reduces NRG3 binding to its receptor to less than 50%,preferably less than 20%, most preferably less than 10% of the bindingof an NRG3 under like conditions.

[0031] In yet another embodiment, the invention includes an agonist of anovel NRG3 of the present invention. The agonist of the invention may bea NRG3, or it may be an anti-NRG3 receptor antibody or receptor bindingfragment. An agonist NRG3 of the invention may also be an polypeptideencoded by an alternatively spliced form of the native NRG3-encodinggene, preferably comprising the NRG3 EGF-like domain disclosed herein.In an embodiment of the agonist of the invention, the NRG3 agonist is ananti-ErbB4 receptor antibody, which antibody binds to and activates theErbB4 receptor. Preferably, the binding affinity of the agonist is atleast 25% of the affinity of the native ligand, more preferably at least50%, and most preferably at least 90% of the affinity of the nativeligand. Similarly, it is preferred that the agonist of the inventionactivate the ErbB4 receptor at the level of at least 25%, morepreferably at least 50%, most preferably at least 90% of activation ofthe native NRG3.

[0032] The invention further concerns a nucleic acid molecule encoding anovel NRG3 of the present invention, vectors containing such nucleicacid, and host cells transformed with the vectors. The nucleic acidpreferably encodes at least the EGF-like domain of a native or variantErbB4 receptor-specific NRG3 of the present invention. The inventionfurther includes nucleic acids hybridizing under stringent conditions tothe complement of a nucleic acid encoding a native ErbB4receptor-specific NRG3 of the present invention, and encoding a proteinretaining the qualitative ErbB4 receptor-specific binding properties ofa native NRG3 disclosed herein. In addition, the invention includes anucleic acid deposited with the American Type Culture Collection as ATCC209156 (pLXSN.mNRG3), which nucleic acid is an expression vectorcomprising nucleic acid encoding the mouse NRG3 open reading frame (SEQID NO: 1). The invention also includes a nucleic acid deposited with theAmerican Type Culture Collection as ATCC 209157 (pRK5.tk.neo.hNRG3B1),which nucleic acid is an expression vector comprising nucleic acidencoding a human NRG3 nucleic acid (SEQ ID NO: 5). The invention alsoincludes a nucleic acid deposited with the American Type CultureCollection as ATCC 209297 (pRK5.tk.neo.hNRG3B2), which nucleic acid isan expression vector comprising nucleic acid encoding an alternativelyspliced form of human NRG3 nucleic acid (SEQ ID NO: 22) lacking nucleicacids 1585 to 1656 of SEQ ID NO: 5. The deduced amino acid sequence ofthe alternatively spliced human NRG3B2 is found in SEQ ID NO: 23 whichlacks amino acids 529 to 552 of SEQ ID NO: 6. A comparison of thehNRG3B1 and hNRG3B2 amino acid sequences is shown in FIG. 4B. Theinvention further includes NRG3 amino acid sequences of mouse and humanNRG3, alternatively spliced forms or fragments thereof, encoded by thedeposited expression vectors.

[0033] In another aspect, the invention concerns a process for producinga NRG3 of the invention, which process comprises transforming a hostcell with nucleic acid encoding the desired NRG3, culturing thetransformed host cell and recovering the NRG3 produced from the hostcell or host cell culture.

[0034] As an alternative to production of the NRG3 in a transformed hostcell, the invention provides a method for producing NRG3 comprising: (a)transforming a cell containing an endogenous NRG3 gene with a homologousDNA comprising an amplifiable gene and a flanking sequence of at leastabout 150 base pairs that is homologous with a DNA sequence within or inproximity to the endogenous NRG3 gene, whereby the homologous DNAintegrates into the cell genome by recombination; (b) culturing the cellunder conditions that select for amplification of the amplifiable gene,whereby the NRG3 gene is also amplified; and thereafter (c) recoveringNRG3 from the cell.

[0035] In a further aspect, the invention concerns an antibody thatbinds specifically to a NRG3 of the present invention, and to ahybridoma cell line producing such an antibody.

[0036] In a still further aspect, the invention concerns animmunoadhesin comprising a novel NRG3 sequence, as disclosed herein,fused to an immunoglobulin sequence. The NRG3 sequence is preferably atransmembrane-domain-deleted form of a native or variant polypeptidefused to an immunoglobulin constant domain sequence, and comprises atleast the EGF-like domain of the extracellular domain of a native NRG3of the present invention. In another preferred embodiment, the NRG3sequence present in the immunoadhesin shows at least about 80% sequencehomology with the extracellular domain of the sequence shown in SEQ IDNO: 3 NRG3 or SEQ ID NO: 7 for mouse or human NRG3, respectively. Theimmunoglobulin constant domain sequence preferably is that of an IgG-1,IgG-2 or IgG-3 molecule, but may also be an IgA or IgM molecule.

[0037] In a further aspect, the invention encompasses a transgenicanimal comprising an altered NRG3 gene in which the polypeptide encodedby the altered gene is not biologically active (non-functional),deleted, or has no more than 70% wild type activity, preferably no morethat 50% activity and more preferably no more than 25% activity of thenative NRG3 polypeptide. In addition, a transgenic animal of theinvention includes a transgenic animal comprising and expressing anative NRG3, alternatively spliced form of NRG3, or a fragment orvariant thereof. Such transgenic animals are useful for the screening ofpotential NRG3 agonists and antagonists.

[0038] The invention further concerns pharmaceutical compositionscomprising a NRG3 as hereinabove defined in admixture with apharmaceutically acceptable carrier. Dosages and administration of NRG3in a pharmaceutical composition may be determined by one of ordinaryskill in the art of clinical pharmacology or pharmacokinetics (see, forexample, Mordenti, J. and Rescigno, A. (1992) Pharmaceutical Research 9:17-25; Morenti, J. et al. (1991) Pharmaceutical Research 8:1351-1359;and Mordenti, J. and Chappell, W. (1989) “The use of interspeciesscaling in toxicokinetics” in Toxicokinetics and New Drug Development,Yacobi et al. (eds), Pergamon Press, NY, pp. 42-96, each of whichreferences are herein incorporated by reference in its entirety).

[0039] In an aspect of the invention, the isolated nucleic acid encodingthe NRG3 of the invention, or fragment thereof, may also be used for invivo or ex vivo gene therapy.

[0040] In an embodiment of the invention, a nucleic acid sequenceencoding an NRG3, or fragment or variant thereof, is introduced into acell of an animal as part of an expression cassette such that theNRG3-encoding nucleic acid sequence is expressed in the cell.Preferably, the NRG3 encoding nucleic acid sequence comprises sequences(such as a promotor sequence) for the control of NRG3 expression withinthe cell. Preferably, the expression cassette comprises a retroviralvector for delivery of the nucleic acid sequence to a cell of theanimal.

[0041] In a further embodiment of the invention, a host cell expressingan NRG3 or NRG3 agonist is introduced into an animal, preferably ahuman, such that NRG3 or NRG3 agonist produced by the host cell iseffective in treating a disorder responsive to increased local orsystemic NRG3 administration. Cells genetically engineered to express anNRG3, fragment or variant thereof, can be implanted in the host toprovide effective levels of factor or factors. The cells can beprepared, encapsulated, and implanted as provided in U.S. Pat. No.4,892,538, and 5,011,472, WO 92/19195, WO 95/05452, or Aebischer et al.(1996) Nature Medicine 2:696-699, for example, which references areherein incorporated by reference in their entirety.

[0042] The present invention includes methods of enhancing survival,proliferation or differentiation of cells comprising the ErbB4 receptorin vivo and in vitro. Normally, the cells will be treated with the NRG3polypeptide or fragment or variant thereof. However, gene therapyapproaches have been described in the art and are encompassed by thepresent invention. These techniques include gene delivery to a cellusing adenovirus, herpes simplex I virus or adeno-associated virus aswell as lipid-based delivery systems (e.g. liposomes). Retroviruses areuseful for ex vivo gene therapy approaches. Accordingly, it is possibleto administer the nucleic acid encoding NRG3, resulting in expression ofthe NRG3 polypeptide, fragment or variant in the patient or in tissueculture. For exemplary gene therapy techniques see WO 93/25673 and thereferences cited therein.

[0043] An aspect of the invention is a method of treating a disorder byadministering to a mammal a cell encoding an NRG3 or fragment thereof,or agonist or antagonist of the NRG3 as necessary to treat the disorder,which cell secretes the NRG3 of the invention. An embodiment of theinvention is a method for preventing or treating damage to a nerve ordamage to other NRG3-expressing or NRG3-responsive cells, e.g. brain,heart, or kidney cells, which method comprises implanting cells thatsecrete NRG3, or fragment or agonist thereof, or antagonist as may berequired for the particular condition, into the body of patients in needthereof.

[0044] A further embodiment of the invention includes an implantationdevice, for preventing or treating nerve damage or damage to other cellsas taught herein, containing a semipermeable membrane and a cell thatsecretes NRG3, or fragment or agonist thereof, (or antagonist as may berequired for the particular condition) encapsulated within the membrane,the membrane being permeable to NRG3, or fragment agonist thereof, andimpermeable to factors from the patient detrimental to the cells. Thepatient's own cells, transformed to produce NRG3 ex vivo, could beimplanted directly into the patient, optionally without suchencapsulation. The methodology for the membrane encapsulation of livingcells is familiar to those of ordinary skill in the art, and thepreparation of the encapsulated cells and their implantation in patientsmay be accomplished readily as is known in the art.

[0045] In accordance with the in vitro methods of the invention, cellscomprising the ErbB4 receptor are placed in a cell culture medium.Examples of ErbB4-receptor-containing cells include neural cells, e.g.,brain cells (such as neurons of the neocortex, cerebellum andhippocampus); cardiac cells; skeletal and smooth muscle cells; andcultured cells transformed with a recombinant NRG3.

[0046] Suitable tissue culture media are well known to persons skilledin the art and include, but are not limited to, Minimal Essential Medium(MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). Thesetissue culture medias are commercially available from Sigma ChemicalCompany (St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells arethen cultured in the cell culture medium under conditions sufficient forthe cells to remain viable and grow in the presence of an effectiveamount of NRG3. The cells can be cultured in a variety of ways,including culturing in a clot, agar, or liquid culture.

[0047] The cells are cultured at a physiologically acceptabletemperature such as 37° C., for example, in the presence of an effectiveamount of NRG3, fragment or variant. The amount of NRG3 may vary, butpreferably is in the range of about 0.1 ng/ml to about 1 mg/mlpreferably about 0.1 ng/ml to about 0.1 ng/ml. The NRG3 can of course beadded to the culture at a dose determined empirically by those in theart without undue experimentation. The concentration of NRG3 in theculture will depend on various factors, such as the conditions underwhich the cells and NRG3 are cultured. The specific temperature andduration of incubation, as well as other culture conditions, can bevaried depending on such factors as, e.g., the concentration of theNRG3, and the type of cells and medium. Those skilled in the art will beable to determine operative and optimal culture conditions without undueexperimentation. Proliferation, differentiation and/or survival of thecells (e.g. neurons) in the cultures can be determined by various assaysknown in the art such as those described above.

[0048] It is contemplated that using NRG3 to enhance cell survival,growth and/or differentiation in vitro will be useful in a variety ofways. For instance, neural cells cultured in vitro in the presence ofNRG3 can be infused into a mammal suffering from reduced levels of thecells. Stable in vitro cultures can also be used for isolatingcell-specific factors and for expression of endogenous or recombinantlyintroduced proteins in the cell. NRG3, fragments or variants thereof mayalso be used to enhance cell survival, proliferation and/ordifferentiation of cells which support the growth and/or differentiationof other cells in cell culture.

[0049] The invention also provides in vivo uses for NRG3. Based on theneuronal cell expression pattern of NRG3, it is believed that thismolecule will be particularly useful for treating diseases which involveneural cell growth such as demyelination, or damage or loss of glialcells (e.g. multiple sclerosis).

[0050] The invention further provides a method for treating a mammalcomprising administering a therapeutically effective amount of NRG3,NRG3 fragment, or NRG3 agonist to the mammal.

[0051] For example, the mammal may be suffering from a neurological ormuscular disorder. Where the disorder is a neurological disorder, NRG3is believed to be useful in promoting the development, maintenance,and/or regeneration of neurons in vivo, including central (brain andspinal chord), peripheral (sympathetic, parasympathetic, sensory, andenteric neurons), and motoneurons. Accordingly, NRG3 may be utilized inmethods for the diagnosis and/or treatment of a variety of neurologicdiseases or disorders which affect the nervous system of a mammal, suchas a human.

[0052] Such diseases or disorders may arise in a patient in whom thenervous system has been damaged by, e.g., trauma, surgery, stroke,ischemia, infection, metabolic disease, nutritional deficiency,malignancy, or toxic agents. The agent is designed to promote thesurvival or growth of neurons. For example, NRG3 can be used to promotethe survival or growth of motoneurons that are damaged by trauma orsurgery. Also, NRG3 can be used to treat motoneuron disorders, such asamyotrophic lateral sclerosis (Lou Gehrig's disease), Bell's palsy, andvarious conditions involving spinal muscular atrophy, or paralysis. NRG3can be used to treat human “neurodegenerative disorders”, such asAlzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis,Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere'sdisease.

[0053] Further, NRG3 can be used to treat neuropathy, and especiallyperipheral neuropathy. “Peripheral neuropathy” refers to a disorderaffecting the peripheral nervous system, most often manifested as one ora combination of motor, sensory, sensorimotor, or autonomic neuraldysfunction. The wide variety of morphologies exhibited by peripheralneuropathies can each be attributed uniquely to an equally wide numberof causes. For example, peripheral neuropathies can be geneticallyacquired, can result from a systemic disease, or can be induced by atoxic agent. Examples include but are not limited to distal sensorimotorneuropathy, or autonomic neuropathies such as reduced motility of thegastrointestinal tract or atony of the urinary bladder. Examples ofneuropathies associated with systemic disease include post-poliosyndrome; examples of hereditary neuropathies includeCharcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia,Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry'sdisease, and Dejerine-Sottas syndrome; and examples of neuropathiescaused by a toxic agent include those caused by treatment with achemotherapeutic agent such as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine.

[0054] The invention further provides a method for treating a mammalcomprising administering a therapeutically effective amount of a NRG3antagonist to the mammal. The mammal in this latter case is one whichcould benefit from a reduction in NRG3 levels/biological activity.

[0055] These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the structure, synthesis, and usage as more fullyset forth below. Each reference cited herein is herein incorporated byreference in its entirety with particular attention to the descriptionof subject matter associated with the context of the citation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 shows the nucleic acid coding sequence of mouse NRG3 cDNA(mNRG3, SEQ ID NO: 1) in which the start (ATG) and stop (TGA) codons ofthe coding sequence are indicated by underlining.

[0057]FIG. 2 shows the nucleic acid coding sequence of human NRG3 cDNA(hNRG3B1, SEQ ID NO: 5) in which the start (ATG) and stop (TGA) codonsof the coding sequence are indicated by underlining.

[0058]FIG. 3 shows the nucleic acid coding sequence of an alternativelyspliced form of human NRG3 cDNA (hNRG3B2; SEQ ID NO: 22) in which thestart (ATG) and stop (TGA) codons of the coding sequence are indicatedby underlining.

[0059] FIGS. 4A-4B.

[0060]FIG. 4A shows the deduced amino acid sequences from mouse (mNRG3)and human (hNRG3B1) cDNA as shown in FIGS. 1 and 2. Mouse NRG3 deducedamino acid sequence is depicted by SEQ ID NO: 2 and human NRG3B 1deduced amino acid sequence is depicted by SEQ ID NO: 6. Variousputative domains within the amino acid sequences are shown. The EGF-likedomain, the N-terminal hydrophobic segment (double underline), theserine/threonine-rich portion, and a predicted transmembrane domain(single underline) are highlighted.

[0061]FIG. 4B shows the deduced amino acid sequences from hNRG3B 1 andhNRG3B2 cDNA as shown in FIGS. 2 and 3. Human NRGB 1 deduced amino acidsequence is depicted by SEQ ID NO: 6 and human NRG3B2 deduced amino acidsequence is depicted by SEQ ID NO: 23. The region of the NRG3 amino acidsequence that differs between the two human sequences is illustrated.

[0062]FIG. 5 shows a sequence alignment of the EGF-like domains of humanNRG3B1 (hNRG3.egf; SEQ ID NO: 4); chicken ARIA (cARIA.egf; SEQ ID NO:9); human amphiregulin (hAR.egf; SEQ ID NO: 10); human betacellulin(hBTC.egf; SEQ ID NO: 1); human EGF (hEGF.egf; SEQ ID NO: 12); humanheparin-binding EGF-like growth factor (hHB-EGF.egf; SEQ ID NO: 13);human heregulin-α (hHRGα; SEQ ID NO: 14); human heregulin-β (hHRGβ.egf;SEQ ID NO: 15); human TGF-α (hTGFα.egf; SEQ ID NO: 16); and mouseepiregulin (mEPR.egf; SEQ ID NO: 17). The sequences were analyzed usingSequence Analysis Programs, Genentech, Inc.

[0063]FIG. 6A-6H are FACS plots demonstrating binding of NRG3^(EGF).Fcto ErbB4 receptor expressed on the surface of cells. In FIGS. 6A-6D,parental K562 cells (FIG. 6A) or K562 cells expressing either ErbB2receptor (K562^(erbB2) cells; FIG. 6B), ErbB3 receptor (K562^(erbB)3cells; FIG. 6C) or ErbB4 receptor (K562^(erbB)4 cells; FIG. 6D) wereexamined for the expression of corresponding receptors. Cells wereincubated with anti-ErbB2 receptor, anti-ErbB3 receptor or anti-ErbB4receptor antibodies as indicated before PE-conjugated secondary antibodywas added. “LOG PE” represents relative fluorescent intensity and“Counts” represents cell numbers. In FIGS. 6E-6H, NRG3^(EGF).Fc is shownby FACS analysis to bind to ErbB4 receptor expressing cells. ParentalK562 cells (FIG. 6E), K562^(erbB2) cells (FIG. 6F), K562^(erbB3) cells(FIG. 6G) and K562^(erbB4) cells (FIG. 6H) were incubated with orwithout NRG3^(EGF).Fc (containing gD tag) for 1 hour, followed byanti-gD-tag primary antibody and PE-conjugated secondary antibody.

[0064]FIG. 7 is a graphical analysis showing competitive inhibition of¹²⁵I-NRG3_(EGF).Fc binding to immobilized soluble ErbB4 receptor byNRG3^(EGF).Fc or NRG^(EGF). Soluble ErbB4 receptor was immobilized on96-well plates, and was incubated with various concentrations ofunlabeled NRG3^(EGF).Fc or NRG^(EGF) and constant amount of ¹²⁵I-labeledNRG3^(EGF).Fc for 1.5 hour at room temperature. The fraction ofradioactivity bound over total ¹²⁵I-NRG3^(EGF).Fc input is plottedagainst the concentration of competitor. Data of a representativeexperiment from four independent assays is shown. Error bars indicatestandard deviation of quadruplicate samples.

[0065] Before the present polypeptides, nucleic acids, vectors, and hostcells and processes for making such are described, it is to beunderstood that this invention is not limited to the particularcompositions of matter and processes described, as such compounds andmethods may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting since the scope ofthe present invention will be limited only by the appended claims.

DESCRIPTION OF THE EMBODIMENTS

[0066] Definitions

[0067] The phrases “novel neuregulin related ligand”, “novel NRG3”,“novel ErbB4 receptor-specific NRG3” are used interchangeably and referto a new member of the family of neuregulins, which NRG3 is expressedspecifically in the brain and nervous system of the embryo and adults,and to functional derivatives of such native polypeptides.

[0068] The term “NRG3” or “neuregulin related ligand” is defined hereinto be any polypeptide sequence that possesses at least one biologicalproperty (as defined below) of native amino acid sequence NRG3 of SEQ IDNO: 2 or 6 (mouse or human, respectively) and additionally includes analternatively spliced form of human NRG3 having the amino acid sequenceof SEQ ID NO: 23. This definition encompasses not only the polypeptideisolated from a native NRG3 source such as human MDA-MB-175 cells orfrom another source, such as another animal species or alternativelyspliced forms of NRG3, but also the polypeptide prepared by recombinantor synthetic methods. It also includes variant forms includingfunctional derivatives, allelic variants, naturally occurring isoformsand analogues thereof. Sometimes the NRG3 is “native NRG3” which refersto endogenous NRG3 polypeptide which has been isolated from a mammal.The NRG3 can also be “native sequence NRG3” insofar as it has the sameamino acid sequence as a native NRG3 (e.g. mouse (SEQ ID NO: 2) or human(SEQ ID NO: 6 or SEQ ID NO: 23) NRG3 shown in FIGS. 4A and 4B). However,“native sequence NRG3” encompasses the polypeptide produced byrecombinant or synthetic means. “Mature NRG3” is soluble or secretedNRG3 released from the cell (i.e. lacking an N-terminal hydrophobicsequence). In this context, NRG3 refers to novel NRG3s comprising anEGF-like domain within an extracellular domain, a transmembrane domainand a cytoplasmic domain, with or without a native signal sequence, andnaturally occurring soluble forms of such NRG3s, with or without theinitiating methionine, whether purified from native source, synthesized,produced by recombinant DNA technology or by any combination of theseand/or other methods. The native NRG3s of the present inventionspecifically include the murine NRG3, the amino acid sequence of whichis shown in FIG. 4 (SEQ. ID. NO: 2), and the human NRG3s having theamino acid sequences shown in FIG. 4 (SEQ. ID. NO: 6 or SEQ ID NO: 23),and fragments or mammalian homologues or alternatively spliced forms ofthese native ligands. The novel native murine and human NRG3s of thepresent invention are about 713 and 720 amino acids in length,respectively, and comprise an EGF-like domain, the N-terminalhydrophobic segment, the serine/threonine-rich portion, a predictedtransmembrane domain, and a predicted intracellular domain. Theboundaries of these domain are indicated in FIG. 4 for the novel murineand human NRG3 sequences.

[0069] Optionally, the NRG3 is not associated with native glycosylation.“Native glycosylation” refers to the carbohydrate moieties which arecovalently attached to native NRG3 when it is produced in the mammaliancell from which the native NRG3 is derived. Accordingly, human NRG3produced in a non-human could be described as not being associated withnative glycosylation, for example it may be glycosylated other than thenative glycosylation. Sometimes, the NRG3 is not associated with anyglycosylation whatsoever (e.g. as a result of being producedrecombinantly in a prokaryote).

[0070] The term “EGF-like domain” refers to an extracellular epidermalgrowth factor (EGF)-like domain of a polypeptide, preferably a NRG3polypeptide of the invention. The EGF-like domain is sufficient to bindneuregulin receptors and stimulate cellular responses (Holmes, W. E., etal. (1992) Science 256:1205-1210). Preferably, an EGF-like domain of theNRG3 of the invention has the amino acid sequence of the NRG3s shown inSEQ ID NO: 4 (mouse or human NRG3 EGF-like domain), where the EGF-likedomain is from about amino acid 284 to about amino acid 332 of humanNRG3, and from about amino acid 286 to about amino acid 334 of mouseNRG3. The NRG3 of the invention encompasses a polypeptide encoded by analternatively spliced form the NRG3 encoding gene, which alternativelyspliced form comprises the NRG3 EGF-like domain.

[0071] The term “ErbB” when used herein refers to any one or more of themammalian ErbB receptors (i.e. ErbB1 or epidermal growth factor (EGF)receptor; ErbB2 or HER2 receptor; ErbB3 or HER3 receptor; ErbB4 or HER4receptor; and any other member(s) of this class I tyrosine kinase familyto be identified in the future) and “erbB” refers to the mammalian erbBgenes encoding these receptors.

[0072] The terms “soluble form”, “soluble receptor”, “soluble NRG3”,“soluble NRG3”, and grammatical variants thereof, refer to variants ofthe native or variant NRG3s of the present invention which are devoid ofa functional transmembrane domain. In the soluble receptors thetransmembrane domain may be deleted, truncated or otherwise inactivatedsuch that they are not capable of cell membrane anchorage. If desired,such soluble forms of the NRG3s of the present invention mightadditionally have their cytoplasmic domains fully or partially deletedor otherwise inactivated.

[0073] A “functional derivative” of a polypeptide is a compound having aqualitative biological activity in common with the native polypeptide.Thus, a functional derivative of a native novel NRG3 of the presentinvention is a compound that has a qualitative biological activity incommon with such native NRG3. “Functional derivatives” include, but arenot limited to, fragments of native polypeptides from any animal species(including humans), derivatives of native (human and non-human)polypeptides and their fragments, and peptide and non-peptide analogs ofnative polypeptides, provided that they have a biological activity incommon with a respective native polypeptide.

[0074] As used herein, the term “fragments” refers to regions within thesequence of a mature native polypeptide. Preferably NRG3 fragments willhave a consecutive sequence of at least 20, and more preferably at least50, amino acid residues of the EGF-like domain of NRG3. The preferredfragments have about 30-150 amino acid residues which are identical to aportion of the sequence of NRG3 in SEQ ID NO: 2 (from mouse), or in SEQID NO: 6 or SEQ ID NO: 23 (from human). The term “derivative” is used todefine amino acid sequence and glycosylation variants, and covalentmodifications of a native polypeptide. “Non-peptide analogs” are organiccompounds which display substantially the same surface as peptideanalogs of the native polypeptides. Thus, the non-peptide analogs of thenative novel NRG3s of the present invention are organic compounds whichdisplay substantially the same surface as peptide analogs of the nativeNRG3s. Such compounds interact with other molecules in a similar fashionas the peptide analogs, and mimic a biological activity of a native NRG3of the present invention. Preferably, amino acid sequence variants ofthe present invention retain at least one domain of a native NRG3,preferably an EGF-like domain, or have at least about 60% amino acidsequence identity, more preferably at least about 75% amino acidsequence identity, and most preferably at least about 90% amino acidsequence identity with a domain of a native NRG3 of the presentinvention. The amino acid sequence variants preferably show the highestdegree of amino acid sequence homology with the EGF-like domain ofnative NRG3s of the present invention. These are the domains which showthe highest percentage amino acid conservation between the novel NRG3sof the present invention and other members of the NRG3 family (see FIG.4).

[0075] The terms “isolated” or “substantially pure” refer to apolypeptide or nucleic acid which is free of other polypeptides ornucleic acids as well as lipids, carbohydrates or other materials withwhich it is naturally associated. An exception is made for glycosylationwherein sugar moieties are covalently attached to amino acids of theNRG3 polypeptide of the invention. One of ordinary skill in the art canpurify a NRG3 polypeptide or nucleic acid encoding the polypeptide usingstandard techniques appropriate for each type of molecule.

[0076] The term “percent amino acid sequence identity” with respect tothe NRG3 sequence is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesin the NRG3 sequence having the deduced amino acid sequence described inFIG. 1, after aligning the sequences and introducing gaps, if necessary,to achieve the maximum percent sequence identity, and not consideringany conservative substitutions as part of the sequence identity.N-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the NRG3 sequence shall be construed as affecting sequence identityor homology.

[0077] Another type of NRG3 variant is “chimeric NRG3 ”, which termencompasses a polypeptide comprising full-length NRG3 or a fragmentthereof fused or bonded to a heterologous polypeptide. The chimera willnormally share at least one biological property with NRG3. Examples ofchimeric NRG3s include immunoadhesins and epitope tagged NRG3. Inanother embodiment, the heterologous polypeptide is thioredoxin, asalvage receptor binding epitope, cytotoxic polypeptide or enzyme (e.g.,one which converts a prodrug to an active drug).

[0078] The terms “covalent modification” and “covalent derivatives” areused interchangeably and include, but are not limited to, modificationsof a native polypeptide or a fragment thereof with an organicproteinaceous or non-proteinaceous derivatizing agent, fusions toheterologous polypeptide sequences, and post-translationalmodifications. Covalent modifications are traditionally introduced byreacting targeted amino acid residues with an organic derivatizing agentthat is capable of reacting with selected sides or terminal residues, orby harnessing mechanisms of post-translational modifications thatfunction in selected recombinant host cells. Certain post-translationalmodifications are the result of the action of recombinant host cells onthe expressed polypeptide. Glutaminyl and asparaginyl residues arefrequently post-translationally deamidated to the corresponding glutamyland aspartyl residues. Alternatively, these residues are deamidatedunder mildly acidic conditions. Other post-translational modificationsinclude hydroxylation of proline and lysine, phosphorylation of hydroxylgroups of seryl, tyrosyl or threonyl residues, methylation of thea-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton (1983) Proteins: Structure and Molecular Properties, W. H.Freeman & Co., San Francisco, pp. 79-86). Covalentderivatives/modifications specifically include fusion proteinscomprising native NRG3 sequences of the present invention and theiramino acid sequence variants, such as immunoadhesins, and N-terminalfusions to heterologous signal sequences.

[0079] The term “biological activity” in the context of the presentinvention is defined as the possession of at least one adhesive,regulatory or effector function qualitatively in common with a nativepolypeptide. Preferred functional derivatives within the scope of thepresent invention are unified by retaining an EGF-like domain and ErbB4receptor-specific binding of a native NRG3 of the present invention.

[0080] The phrase “activating an ErbB receptor” refers to the act ofcausing the intracellular kinase domain of an ErbB receptor tophosphorylate tyrosine residues. Generally, this will involve binding ofNRG3 to an ErbB4 receptor or ErbB4 receptor homodimer, which bindingactivates a kinase domain of one or more of the receptors and therebyresults in phosphorylation of tyrosine residues in one or more of thereceptors, and/or phosphorylation of tyrosine residues in additionalsubstrate polypeptide(s). ErbB receptor phosphorylation can bequantified using the tyrosine phosphorylation assays described below. Itis understood that the NRG3 of the invention may itself be activated byinteraction with an ErbB receptor via the intracellular domain of NRG3.Thus, an NRG3-activating ligand that binds to the NRG3 (preferablybinding to the extracellular domain, more preferably the EGF-likedomain) includes, but is not limited to, a ligand, an antibody, or areceptor. Activation of the NRG3 may be through phosphorylation of theintracellular domain or other like event common to receptor/ligandmediated cell signaling. As a mediator of cell signaling, the NRG3 ofthe invention is expected to be associated with apoptosis, metabolicsignaling, differentiation or cell proliferation.

[0081] “Identity” or “homology” with respect to a native polypeptide andits functional derivative is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with theresidues of a corresponding native polypeptide, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent homology, and not considering any conservative substitutions aspart of the sequence identity. Neither N- or C-terminal extensions norinsertions shall be construed as reducing identity or homology. Methodsand computer programs for the alignment are well known in the art. Forexample, the sequences disclosed herein were analyzed using SequenceAnalysis Programs, Genentech, Inc, Inc.

[0082] The term “agonist” is used to refer to peptide and non-peptideanalogs of the native NRG3s of the present invention and to antibodiesspecifically binding such native NRG3s provided that they retain atleast one biological activity of a native NRG3. Preferably, the agonistsof the present invention retain the qualitative EGF-like domain bindingrecognition properties of the native NRG3 polypeptides.

[0083] The term “antagonist” is used to refer to a molecule inhibiting abiological activity of a native NRG3 of the present invention.Preferably, the antagonists herein inhibit the binding of a native NRG3of the present invention. Preferred antagonists essentially completelyblock the binding of a native NRG3 to an ErbB4 receptor to which itotherwise binds. A NRG3 “antagonist” is a molecule which prevents, orinterferes with, a NRG3 effector function (e.g. a molecule whichprevents or interferes with binding and/or activation of an ErbB4receptor by NRG3). Such molecules can be screened for their ability tocompetitively inhibit ErbB receptor activation by NRG3 in the tyrosinephosphorylation assay disclosed herein, for example. Preferredantagonists are those which do not substantially interfere with theinteraction of other heregulin polypeptides with ErbB receptor(s).Examples of NRG3 antagonists include neutralizing antibodies againstNRG3 and antisense polynucleotides against the NRG3 gene.

[0084] Ordinarily, the terms “amino acid” and “amino acids” refer to allnaturally occurring L-α-amino acids. In some embodiments, however,D-amino acids may be present in the polypeptides or peptides of thepresent invention in order to facilitate conformational restriction. Forexample, in order to facilitate disulfide bond formation and stability,a D amino acid cysteine may be provided at one or both termini of apeptide functional derivative or peptide antagonist of the native NRG3sof the present invention. The amino acids are identified by either thesingle-letter or three-letter designations: Asp D aspartic acid Ile Iisoleucine Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine GluE glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly Gglycine Lys K lysine Ala A alanine Arg R arginine Cys C cysteine Trp Wtryptophan Val V valine Gln Q glutamine Met M methionine Asn Nasparagine

[0085] The term “amino acid sequence variant” refers to molecules withsome differences in their amino acid sequences as compared to a nativeamino acid sequence.

[0086] Substitutional variants are those that have at least one aminoacid residue in a native sequence removed and a different amino acidinserted in its place at the same position.

[0087] Insertional variants are those with one or more amino acidsinserted immediately adjacent to an amino acid at a particular positionin a native sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

[0088] Deletional variants are those with one or more amino acids in thenative amino acid sequence removed.

[0089] “Antibodies (Abs)” and “immunoglobulins (Igs)” are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

[0090] Native antibodies and immunoglobulins are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies between the heavy chainsof different immunoglobulin isotypes. Each heavy and light chain alsohas regularly spaced intrachain disulfide bridges.

[0091] Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one and (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains (Clothia et al. (1985) J. Mol. Biol. 186,651-663; Novotny and Haber (1985) Proc. Natl. Acad. Sci. USA82:4592-4596).

[0092] The light chains of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda (X), based on the amino acid sequences of theirconstant domains.

[0093] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called a, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

[0094] The term “antibody” is used in the broadest sense andspecifically covers single monoclonal antibodies (including agonist andantagonist antibodies), antibody compositions with polyepitopicspecificity, as well as antibody fragments (e.g., Fab, F(ab′)₂, and Fv),so long as they exhibit the desired biological activity.

[0095] The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein (1975) Nature 256:495, or may be made by recombinantDNA methods (see, e.g. U.S. Pat. No. 4,816,567 (Cabilly et al.) and Mageand Lamoyi (1987) in Monoclonal Antibody Production Techniques andApplications, pp. 79-97, Marcel Dekker, Inc., New York). The monoclonalantibodies may also be isolated from phage libraries generated using thetechniques described in McCafferty et al. (1990) Nature 348:552-554, forexample.

[0096] “Humanized” forms of non-human (e.g. murine) antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof (such as Fv, Fab, Fab′, F(ab)₂ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. For further details see: Jones et al.(1986) Nature 321:522-525; Reichmann et al. (1988) Nature 332:323-329;EP-B-239 400 published Sep. 30, 1987; Presta (1992) Curr. Op. Struct.Biol. 2:593-596; and EP-B-451 216 published Jan. 24, 1996), whichreferences are herein incorporated by reference in their entirety. Thehumanized antibody includes a Primatized™ antibody wherein theantigen-binding region of the antibody is derived from an antibodyproduced by immunizing macaque monkeys with the antigen of interest.

[0097] By “neutralizing antibody” is meant an antibody molecule asherein defined which is able to block or significantly reduce aneffector function of native sequence NRG3. For example, a neutralizingantibody may inhibit or reduce the ability of NRG3 to activate an ErbBreceptor, preferably an ErbB4 receptor, in the tyrosine phosphorylationassay described herein. The neutralizing antibody may also block themitogenic activity of NRG3 in the cell proliferation assay disclosedherein.

[0098] The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cabilly et al.; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA81:6851-6855).

[0099] In the context of the present invention the expressions “cell”,“cell line”, and “cell culture” and “host cell” are usedinterchangeably, and all such designations include progeny. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological property, as screened for in theoriginally transformed cell, are included. Methods of stable transfer,meaning that the foreign DNA is continuously maintained in the host, areknown in the art.

[0100] The terms “replicable expression vector”, “expression vector” and“vector” refer to a piece of DNA, usually double-stranded, which mayhave inserted into it a piece of foreign DNA. Foreign DNA is defined asheterologous DNA, which is DNA not naturally found in the host cell. Thevector is used to transport the foreign or heterologous DNA into asuitable host cell. Once in the host cell, the vector can replicateindependently of the host chromosomal DNA, and several copies of thevector and its inserted (foreign) DNA may be generated. In addition, thevector contains the necessary elements that permit translating theforeign DNA into a polypeptide. Many molecules of the polypeptideencoded by the foreign DNA can thus be rapidly synthesized.

[0101] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancer.

[0102] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or a secretory leader is operably linked to DNAfor a polypeptide if it is expressed as a preprotein that participatesin the secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, then synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

[0103] “Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods,such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, publishedMay 4, 1988, or via deoxynucleoside H-phosphanate intermediates asdescribed by Froehler et al. (1986) Nucl. Acids Res. 14:5399. They arethen purified on polyacrylamide gels.

[0104] By “solid phase” is meant a non-aqueous matrix to which a reagentof interest (e.g., NRG3 or an antibody thereto) can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149, herein incorporated byreference in its entirety.

[0105] The terms “transformation” and “transfection” are usedinterchangeably herein and refer to the process of introducing DNA intoa cell. Following transformation or transfection, the NRG3 DNA mayintegrate into the host cell genome, or may exist as an extrachromosomalelement. If prokaryotic cells or cells that contain substantial cellwall constructions are used as hosts, the preferred methods oftransfection of the cells with DNA is the calcium treatment methoddescribed by Cohen et al. (1972) Proc. Natl. Acad. Sci. U.S.A.,69:2110-2114 or the polyethylene glycol method of Chung et al. (1988)Nuc. Acids. Res. 16:3580. If yeast are used as the host, transfection isgenerally accomplished using polyethylene glycol, as taught by Hinnen(1978) Proc. Natl. Acad. Sci. U.S.A. 75:1929-1933. If mammalian cellsare used as host cells, transfection generally is carried out by thecalcium phosphate precipitation method, Graham et al. (1978) Virology52:546, Gorman et al. (1990) DNA and Protein Eng. Tech. 2:3-10. However,other known methods for introducing DNA into prokaryotic and eukaryoticcells, such as nuclear injection, electroporation, or protoplast fusionalso are suitable for use in this invention.

[0106] Particularly useful in this invention are expression vectors thatprovide for the transient expression in mammalian cells of DNA encodingNRG3. In general, transient expression involves the use of an expressionvector that is able to efficiently replicate in a host cell, such thatthe host cell accumulates many copies of the expression vector and, inturn, synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties.

[0107] It is further envisioned that the NRG3 of this invention may beproduced by homologous recombination, as provided for in WO 91/06667,published May 16, 1991. Briefly, this method involves transforming acell containing an endogenous NRG3 gene with a homologous DNA, whichhomologous DNA comprises (a) an amplifiable gene (e.g a gene encodingdihydrofolate reductase (DHFR)), and (b) at least one flanking sequence,having a length of at least about 150 base pairs, which is homologouswith a nucleotide sequence in the cell genome that is within or inproximity to the gene encoding NRG3. The transformation is carried outunder conditions such that the homologous DNA integrates into the cellgenome by recombination. Cells having integrated the homologous DNA arethen subjected to conditions which select for amplification of theamplifiable gene, whereby the NRG3 gene is amplified concomitantly. Theresulting cells are then screened for production of desired amounts ofNRG3. Flanking sequences that are in proximity to a gene encoding NRG3are readily identified, for example, by the method of genomic walking,using as a starting point the nucleotide sequence, or fragment thereof,of mouse NRG3 of FIG. 1 (SEQ ID NO: 1), or human NRG3 of FIG. 2 (SEQ IDNO: 5) or FIG. 3 (SEQ ID NO: 22). DNA encoding the mouse and human NRG3polypeptides is deposited with the American Type Culture Collection asATCC 209156 (mouse; pLXSN.mNRG3), ATCC 209157 (human;pRK5.tk.neo.hNRG3B1), or ATCC 209297 (human; pRK5.tk.neo.hNRG3B2).

[0108] The expression “enhancing survival of a cell” refers to the actof increasing the period of existence of a cell, relative to anuntreated cell which has not been exposed to NRG3, either in vitro or invivo.

[0109] The phrase “enhancing proliferation of a cell” encompasses thestep of increasing the extent of growth and/or reproduction of the cell,relative to an untreated cell, either in vitro or in vivo. An increasein cell proliferation in cell culture can be detected by counting thenumber of cells before and after exposure to NRG3 (see the Examplebelow). The extent of proliferation can be quantified via microscopicexamination of the degree of confluency. Cell proliferation can also bequantified by measuring ³H uptake by the cells.

[0110] By “enhancing differentiation of a cell” is meant the act ofincreasing the extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g. identifying morphologicalchanges in the cell).

[0111] “Muscle cells” include skeletal, cardiac or smooth muscle tissuecells. This term encompasses those cells which differentiate to formmore specialized muscle cells (e.g. myoblasts). c“Isolated NRG3 nucleicacid” is RNA or DNA free from at least one contaminating source nucleicacid with which it is normally associated in the natural source andpreferably substantially free of any other mammalian RNA or DNA. Thephrase “free from at least one contaminating source nucleic acid withwhich it is normally associated” includes the case where the nucleicacid is present in the source or natural cell but is in a differentchromosomal location or is otherwise flanked by nucleic acid sequencesnot normally found in the source cell. An example of isolated NRG3nucleic acid is RNA or DNA that encodes a biologically active NRG3sharing at least 75%, more preferably at least 80%, still morepreferably at least 85%, even more preferably 90%, and most preferably95% sequence identity with the mouse NRG3 shown in FIG. 1 (SEQ ID NO:1), or human NRG3 shown in FIG. 2 (SEQ ID NO: 4) or FIG. 3 (SEQ ID NO:22).

[0112] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0113] Hybridization is preferably performed under “stringentconditions” which means (1) employing low ionic strength and hightemperature for washing, for example, 0.015 sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C., or (2) employingduring hybridization a denaturing agent, such as formamide, for example,50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1%SDS. Yet another example is hybridization using a buffer of 10% dextransulfate, 2× SSC (sodium chloride/sodium citrate) and 50% formamide at55° C., followed by a high-stringency wash consisting of 0.1× SSCcontaining EDTA at 55° C.

[0114] “Immunoadhesins” or “NRG3- immunoglobulin chimeras” are chimericantibody-like molecules that combine the functional domain(s) of abinding protein (usually a receptor, a cell-adhesion molecule or aligand) with the an immunoglobulin sequence. The most common example ofthis type of fusion protein combines the hinge and Fc regions of animmunoglobulin (Ig) with domains of a cell-surface receptor thatrecognizes a specific ligand. This type of molecule is called an“immunoadhesin”, because it combines “immune” and “adhesion” functions;other frequently used names are “Ig-chimera”, “Ig-” or “Fc-fusionprotein”, or “receptor-globulin. ”

[0115] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures. those in need of treatment include thosealready with the disorder as well as those prone to have the disorder ofthose in which the disorder is to be prevented.

[0116] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows,and the like. Preferably, the mammal herein is a human.

[0117] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, polyethylene glycol (PEG), and Pluronics™.

[0118] General Procedures for the Production of an NRG3 by RecombinantDNA Technology

[0119] A. Identification and Isolation of Nucleic Acid Encoding NovelNeuregulin Related Ligand, NRG3.

[0120] The native NRG3s of the present invention may be isolated fromcDNA or genomic libraries. For example, a suitable cDNA library can beconstructed by obtaining polyadenylated mRNA from cells known to expressthe desired NRG3, and using the mRNA as a template to synthesize doublestranded cDNA. Suitable sources of the mRNA are embryonic and adultmammalian tissues. mRNA encoding native NRG3s of the present inventionis expressed, for example, in adult mammalian, brain, nervous system,heart, muscle, and testis. The gene encoding the novel NRG3s of thepresent invention can also be obtained from a genomic library, such as ahuman genomic cosmid library, or a mouse-derived embryonic stem cell(ES) genomic library.

[0121] Libraries, either cDNA or genomic, are screened with probesdesigned to identify the gene of interest or the protein encoded by it.For cDNA expression libraries, suitable probes include monoclonal andpolyclonal antibodies that recognize and specifically bind to a NRG3 ofthe invention. For cDNA libraries, suitable probes include carefullyselected oligonucleotide probes (usually of about 20-80 bases in length)that encode known or suspected portions of a NRG3 polypeptide from thesame or different species, and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, 1989, herein incorporated by referencein its entirety.

[0122] If DNA encoding a NRG3 of the present invention is isolated byusing carefully selected oligonucleotide sequences to screen cDNAlibraries from various tissues, the oligonucleotide sequences selectedas probes should be sufficient in length and sufficiently unambiguousthat false positive selections are minimized. The actual nucleotidesequence(s) is/are usually designed based on regions that have the leastcodon redundance. The oligonucleotides may be degenerate at one or morepositions. The use of degenerate oligonucleotides is of particularimportance where a library is screened from a species in whichpreferential codon usage is not known.

[0123] The oligonucleotide must be labeled such that it can be detectedupon hybridization to DNA in the library being screened. The preferredmethod of labeling is to use ATP (e.g., γ³²P) and polynucleotide kinaseto radiolabel the 5′ end of the oligonucleotide. However, other methodsmay be used to label the oligonucleotide, including, but not limited to,biotinylation or enzyme labeling.

[0124] cDNAs encoding the novel NRG3s can also be identified andisolated by other known techniques of recombinant DNA technology, suchas by direct expression cloning, or by using the polymerase chainreaction (PCR) as described in U.S. Pat. No. 4,683,195, issued Jul. 28,1987, in section 14 of Sambrook et al., supra, or in Chapter 15 ofCurrent Protocols in Molecular Biology, Ausubel et al. eds., GreenePublishing Associates and Wiley-Interscience 1991, which references areherein incorporated by reference in their entirety.

[0125] Once cDNA encoding a new native ErbB4 receptor-specific NRG3 fromone species has been isolated, cDNAs from other species can also beobtained by cross-species hybridization. According to this approach,human or other mammalian cDNA or genomic libraries are probed by labeledoligonucleotide sequences selected from known NRG3 sequences (such asmurine or human sequences) in accord with known criteria. Preferably,the probe sequence should be sufficient in length and sufficientlyunambiguous that false positives are minimized. Typically, a ³²P-labeledoligonucleotide having about 30 to 50 bases is sufficient, particularlyif the oligonucleotide contains one or more codons for methionine ortryptophan. Isolated nucleic acid will be DNA that is identified andseparated from contaminant nucleic acid encoding other polypeptides fromthe source of nucleic acid. Hybridization is preferably performed under“stringent conditions”, as defined herein.

[0126] Once the sequence is known, the gene encoding a particular NRG3can also be obtained by chemical synthesis, following one of the methodsdescribed in Engels and Uhlmann, Agnew (1989) Chem. Int. Ed. Engl.28:716, herein incorporated by reference in its entirety. These methodsinclude triester, phosphite, phosphoramidite and H-phosphonate methods,PCR and other autoprimer methods, and oligonucleotide syntheses on solidsupports.

[0127] B. Cloning and Expression of Nucleic Acid Encoding the NovelNRG3s.

[0128] Once the nucleic acid encoding a novel NRG3 is available, it isgenerally ligated into a replicable expression vector for furthercloning (amplification of the DNA), or for expression.

[0129] Expression and cloning vectors are well known in the art andcontain a nucleic acid sequence that enables the vector to replicate inone or more selected host cells. The selection of the appropriate vectorwill depend on 1) whether it is to be used for DNA amplification or forDNA expression, 2) the size of the DNA to be inserted into the vector,and 3) the host cell to be transformed with the vector. Each vectorcontains various components depending on its function (amplification ofDNA of expression of DNA) and the host cell for which it is compatible.The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence. Construction of suitable vectorscontaining one or more of the above listed components, the desiredcoding and control sequences, employs standard ligation techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and religatedin the form desired to generate the plasmids required. For analysis toconfirm correct sequences in plasmids constructed, the ligation mixturesare commonly used to transform E. coli cells, e.g. E. coli K12 strain294 (ATCC 31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al. (1981)Nucleic Acids Res. 9:309 or by the method of Maxam et al. (1980) Methodsin Enzymology 65:499.

[0130] The polypeptides of the present invention may be expressed in avariety of prokaryotic and eukaryotic host cells. Suitable prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. A preferred cloning host is E. coli 294 (ATCC 31,446) althoughother gram negative or gram positive prokaryotes such as E. coli B, E.coli X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonasspecies, or Serratia Marcesans are suitable.

[0131] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable hosts for vectors herein.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species and strains are commonly available and usefulherein, such as S. pombe (Beach and Nurse (1981) Nature 290:140),Kluyveromyces lactis (Louvencourt et al. (1983) J. Bacteriol. 737);yarrowia (EP 402,226); Pichia pastoris (EP 183,070), Trichoderma reesia(EP 244,234), Neurospora crassa (Case et al. (1979) Proc. Nati. Acad.Sci. USA 76:5259-5263); and Aspergillus hosts such as A. nidulans(Ballance et al. (1983) Biochem. Biophys. Res. Commun. 112:284-289;Tilburn et al. (1983) Gene 26:205-221; Yelton et al. (1984) Proc. Natl.Acad. Sci. USA 81:1470-1474) and A. niger (Kelly and Hynes (1985) EMBOJ. 4:475-479).

[0132] Suitable host cells may also derive from multicellular organisms.Such host cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al. (1988) Bio/Technology 6:47-55; Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al. (1985) Nature 315:592-594. Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa califormica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

[0133] Plant cell cultures of cotton, corn, potato, soybean, petunia,tomato, and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the NRG3 DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding a NRG3 is transferred to the plant cellhost such that it is transfected, and will, under appropriateconditions, express the NRG3 DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences.Depicker et al. (1982) J. Mol. Appl. Gen. 1:561. In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published Jun. 21, 1989.

[0134] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) is per sewell known (see for example, Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney cell line (293 or 293 cells subcloned for growthin suspension culture, Graham et al. (1977) J. Gen. Virol. 36:59); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA77:4216); mouse sertolli cells (TM4, Mather (1980) Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad.Sci. 383:44068); MRC 5 cells; FS4 cells; and a human hepatoma cell line(Hep G2). Preferred host cells are human embryonic kidney 293 andChinese hamster ovary cells.

[0135] Particularly useful in the practice of this invention areexpression vectors that provide for the expression in mammalian cells ofDNA encoding a novel NRG3 herein. Where transient expression ispreferred, expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient systems, comprising a suitable expressionvector and a host cell, allow for the convenient positive identificationof polypeptides encoded by cloned DNAs, as well as for the rapidscreening of such polypeptides for desired biological or physiologicalproperties. Thus, transient expression systems are particularly usefulin the invention for purposes of identifying analogs and variants of anative NRG3 of the invention.

[0136] Other methods, vectors, and host cells suitable for adaptation tothe synthesis of the NRG3s in recombinant vertebrate cell culture aredescribed for example, in Getting et al (1981) Nature 293:620-625;Mantel et al. (1979) Nature 281:40-46; Levinson et al.; EP 117,060 andEP 117,058. Particularly useful plasmids for mammalian cell cultureexpression of the NRG3 polypeptides are pRK5 (EP 307,247), or pSVI6B(PCT Publication No. WO 91/08291).

[0137] Other cloning and expression vectors suitable for the expressionof the NRG3s of the present invention in a variety of host cells are,for example, described in EP 457,758 published Nov 27, 1991. A largevariety of expression vectors is now commercially available. Anexemplary commercial yeast expression vector is pPIC.9 (Invitrogen),while an commercially available expression vector suitable fortransformation of E. coli cells is PET15b (Novagen).

[0138] C. Culturing the Host Cells.

[0139] Prokaryote cells used to produced the NRG3s of this invention arecultured in suitable media as describe generally in Sambrook et al.,supra.

[0140] Mammalian cells can be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium (DMEM, Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham and Wallace(1979) Meth. Enzymol. 58:44; Barnes and Sato (1980) Anal. Biochem.102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655;WO 90/03430; WO 87/00195 or U.S. Pat. Re. 30,985 may be used as culturemedia for the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGentamycin™ drug) trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH andthe like, suitably are those previously used with the host cell selectedfor cloning or expression, as the case may be, and will be apparent tothe ordinary artisan.

[0141] The host cells referred to in this disclosure encompass cells inin vitro cell culture as well as cells that are within a host animal orplant.

[0142] It is further envisioned that the NRG3s of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the particular NRG3.

[0143] D. Detecting Gene Amplification and/or Expression.

[0144] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas (1980) Proc.Natl. Acad. Sci. USA 77:5201-5205), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³²p However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

[0145] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. A particularly sensitive staining techniquesuitable for use in the present invention is described by Hse et al.(1980) Am. J. Clin. Pharm. 75:734-738.

[0146] Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native NRG3 polypeptide, or against a synthetic peptide basedon the DNA sequence disclosed herein.

[0147] E. Amino Acid Sequence Variants of a Native NRG3.

[0148] Amino acid sequence variants of native NRG3s are prepared bymethods known in the art by introducing appropriate nucleotide changesinto a native NRG3 DNA, or by in vitro synthesis of the desiredpolypeptide. There are two principal variables in the construction ofamino acid sequence variants: the location of the mutation site and thenature of the mutation. With the exception of naturally-occurringalleles, which do not require the manipulation of the DNA sequenceencoding the native NRG3, the amino acid sequence variants of NRG3s arepreferably constructed by mutating the DNA, either to arrive at anallele or an amino acid sequence variant that does not occur in nature.

[0149] One group of mutations will be created within the extracellulardomain or within the EGF-like domain of a novel native mouse or humanNRG3 of the present invention (see FIG. 3 for the delineation of theextracellular domain (SEQ ID NO: 3 or SEQ ID NO: 7) and EGF-like domain(SEQ ID NO: 4) within human or mouse NRG3 amino acid sequences,respectively. Since these domains are believed to be functionallyimportant, alterations such as non-conservative substitutions,insertions and/or deletions in these regions are expected to result ingenuine changes in the properties of the native receptor molecules suchas in ErbB4 receptor binding and activation. Accordingly, amino acidalterations in this region are also believed to result in variants withproperties significantly different from the corresponding nativepolypeptides. Non-conservative substitutions within these functionallyimportant domains may result in variants which lose the ErbB4 receptorrecognition and binding ability of their native counterparts, or haveincreased ErbB4 receptor recognition properties, enhanced selectivity,or enhanced activation properties as compared to the correspondingnative proteins.

[0150] Alternatively or in addition, amino acid alterations can be madeat sites that differ in novel NRG3s from various species, or in highlyconserved regions, depending on the goal to be achieved. Sites at suchlocations will typically be modified in series, e.g. by (1) substitutingfirst with conservative choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue orresidues, or (3) inserting residues of the same or different classadjacent to the located site, or combinations of options 1-3. Onehelpful technique for such modifications is called “alanine scanning”(Cunningham and Wells (1989) Science 244:1081-1085).

[0151] In yet another group of the variant NRG3s of the presentinvention, one or more of the functionally less significant domains maybe deleted or inactivated. For example, the deletion or inactivation ofthe transmembrane domain yields soluble variants of the native proteins.Alternatively, or in addition, the cytoplasmic domain may be deleted,truncated or otherwise altered.

[0152] Naturally-occurring amino acids are divided into groups based oncommon side chain properties:

[0153] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0154] (2) neutral hydrophobic: cys, ser, thr;

[0155] (3) acidic: asp, glu;

[0156] (4) basic: asn, gln, his, lys, arg;

[0157] (5) residues that influence chain orientation: gly, pro; and

[0158] (6) aromatic: trp, tyr, phe.

[0159] Conservative substitutions involve exchanging a member within onegroup for another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Substantial changes in function or immunological identityare made by NRG3 substitutions that are less conservative, i.e. differmore significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of substitution, for example as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site or (c) the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the properties of the novel native NRG3s of the presentinvention will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. Such substitutions are expected to have their mostsignificant effect when made within the extracellular domain, such as inthe EGF-like domain.

[0160] Substitutional variants of the novel NRG3s of the presentinvention also include variants where functionally homologous (having atleast about 40%-50% homology) domains of other proteins are substitutedby routine methods for one or more of the above-identified domainswithin the novel NRG3 structure, such as the extracellular domain orEGF-like domain.

[0161] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Typically, the transmembrane and cytoplasmic domains, oronly the transmembrane domains are deleted. However, deletion from theC-terminus to any suitable amino acid N-terminal to the transmembraneregion which preserves the biological activity or immunologicalcross-reactivity of a native NRG3 is suitable. The transmembrane region(TM) of each of the human and mouse NRG3 consensus sequences is shown inFIGS. 4A and 4B to range from about amino acid 362 to about amino acid384 (human SEQ ID NO: 6 and SEQ ID NO: 23), and about amino acid 360 toabout amino acid 382 (mouse SEQ ID NO: 2).

[0162] A preferred class of substitutional and/or deletional variants ofthe present invention are those involving a transmembrane region of anovel NRG3 molecule. Transmembrane regions are highly hydrophobic orlipophilic domains that are the proper size to span the lipid bilayer ofthe cellular membrane. They are believed to anchor the NRG3 in the cellmembrane, and allow for homo- or heteropolymeric complex formation.Inactivation of the transmembrane domain, typically by deletion orsubstitution of transmembrane domain hydroxylation residues, willfacilitate recovery and formulation by reducing its cellular or membranelipid affinity and improving its aqueous solubility. If thetransmembrane and cytoplasmic domains are deleted one avoids theintroduction of potentially immunogenic epitopes, whether by exposure ofotherwise intracellular polypeptides that might be recognized by thebody as foreign or by insertion of heterologous polypeptides that arepotentially immunogenic. Inactivation of the membrane insertion functionis accomplished by deletion of sufficient residues to produce asubstantially hydrophilic hydropathy profile in the transmembrane or bysubstituting with heterologous residues which accomplish the sameresult.

[0163] A principle advantage of the transmembrane inactivated variantsof the NRG3s of the present invention is that they may be secreted intothe culture medium of recombinant hosts. These variants are soluble inbody fluids such as blood and do not have an appreciable affinity forcell membrane lipids, thus considerably simplifying their recovery fromrecombinant cell culture. As a general proposition, such solublevariants will retain a functional extracellular domain or fragmentthereof, will not have a functional transmembrane domain, and preferablywill not have a functional cytoplasmic domain.

[0164] For example, the transmembrane domain may be substituted by anyamino acid sequence, e.g. a random or predetermined sequences of about 5to 50 serine, threonine, lysine, arginine, glutamine, aspartic acid andlike hydrophilic residues, which altogether exhibit a hydrophilichydropathy profile. Like the deletional (truncated) soluble variants,these variants are secreted into the culture medium of recombinanthosts.

[0165] Amino acid insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.insertions within the novel NRG3 amino acid sequence) may rangegenerally from about 1 to 10 residues, more preferably 1 to 5 residues,more preferably 1 to 3 residues. An example of a terminal insertionincludes fusion of a heterologous N-terminal signal sequence to theN-terminus of the NRG3 molecule to facilitate the secretion of themature NRG3 or a fragment thereof from recombinant host cells. Suchsignal sequences will generally be obtained from, and thus be homologousto, a signal sequence of the intended host cell species. Suitablesequences include STII or Ipp for E coli, alpha factor for yeast, andviral signals such as herpes gD for mammalian cells.

[0166] Other insertional variants of the native NRG3 molecules includethe fusion of the N- or C-terminus of the NRG3 molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on Apr. 6,1989.

[0167] Further insertional variants are immunologically activederivatives of the novel NRG3s, which comprise the EGF-like domain and apolypeptide containing an epitope of an immunologically competentextraneous polypeptide, i.e. a polypeptide which is capable of elicitingan immune response in the animal to which the fusion is to beadministered or which is capable of being bound by an antibody raisedagainst an extraneous polypeptide. Typical examples of suchimmunologically competent polypeptides are allergens, autoimmuneepitopes, or other potent immunogens or antigens recognized bypre-existing antibodies in the fusion recipient, including bacterialpolypeptides such as trpLE, β-glactosidase, viral polypeptides such asherpes gD protein, and the like.

[0168] Immunogenic fusions are produced by cross-linking in vitro or byculture of cells transformed with recombinant DNA encoding animmunogenic polypeptide. It is preferable that the immunogenic fusion beone in which the immunogenic sequence is joined to or inserted into anovel NRG3 molecule or fragment thereof by one or more peptide bonds.These products therefore consist of a linear polypeptide chaincontaining the NRG3 epitope and at least one epitope foreign to theNRG3. It will be understood that it is within the scope of thisinvention to introduce the epitopes anywhere within a NRG3 molecule ofthe present invention or a fragment thereof. These immunogenicinsertions are particularly useful when formulated into apharmacologically acceptable carrier and administered to a subject inorder to raise antibodies against the NRG3 molecule, which antibodies inturn are useful as diagnostics, in tissue-typing, or in purification ofthe novel NRG3s by standard immunoaffinity techniques. Alternatively, inthe purification of the NRG3s of the present invention, binding partnersfor the fused extraneous polypeptide, e.g. antibodies, receptors orligands, are used to adsorb the fusion from impure admixtures, afterwhich the fusion is eluted and, if desired, the novel NRG3 is recoveredfrom the fusion, e.g. by enzymatic cleavage.

[0169] Since it is often difficult to predict in advance thecharacteristics of a variant NRG3, it will be appreciated that somescreening will be needed to select the optimum variant. Such screeningincludes, but is not limited to, arrays of ErbB4 receptor binding.

[0170] After identifying the desired mutation(s), the gene encoding aNRG3 variant can, for example, be obtained by chemical synthesis asdescribed herein. More preferably, DNA encoding a NRG3 amino acidsequence variant is prepared by site-directed mutagenesis of DNA thatencodes an earlier prepared variant or a nonvariant version of the NRG3.Site-directed (site-specific) mutagenesis allows the production of NRG3variants through the use of specific oligonucleotide sequences thatencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Typically, a primer ofabout 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the techniques of site-specific mutagenesis are well known inthe art, as exemplified by publications such as, Edelman et al. (1983)DNA 2:183. As will be appreciated, the site-specific mutagenesistechnique typically employs a phage vector that exists in both asingle-stranded and double-stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage, forexample, as disclosed by Messing et al., Third Cleveland Symposium onMacromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam(1981). This and other phage vectors are commercially available andtheir use is well known to those skilled in the art. A versatile andefficient procedure for the construction of oligodeoxyribonucleotidedirected site-specific mutations in DNA fragments using Ml 3-derivedvectors was published by Zoller, M. J. and Smith, M. (1982) NucleicAcids Res. 10:6487-6500). Also, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al. (1987) Meth.Enzymol. 153:3) may be employed to obtain single-stranded DNA.Alternatively, nucleotide substitutions are introduced by synthesizingthe appropriate DNA fragment in vitro, and amplifying it by PCRprocedures known in the art.

[0171] The PCR amplification technique may also be used to create aminoacid sequence variants of a novel NRG3. In a specific example of PCRmutagenesis, template plasmid DNA (1 μg) is linearized by digestion witha restriction endonuclease that has a unique recognition site in theplasmid DNA outside of the region to be amplified. Of this material, 100ng is added to a PCR mixture containing PCR buffer, which contains thefour deoxynucleotide triphosphates and is included in the GeneAmp^(R)kits (obtained from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.), and 25 pmole of each oligonucleotide primer, to a final volumeof 50 μl. The reaction mixture is overlayered with 35 μl mineral oil.The reaction is denatured for 5 minutes at 100° C., placed briefly onice, and then 1 μl Thermus aquaticus (Taq) DNA polymerase (5 units/1),purchased from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.) is added below the mineral oil layer. The reaction mixture isthen inserted into a DNA Thermal Cycler (Perkin-Elmer Cetus) programmedas follows: (as an example)

[0172] 2 min. 55° C.,

[0173] 30 sec. 72° C., then 19 cycles of the following:

[0174] 30 sec. 94° C.,

[0175] 30 sec. 55° C., and

[0176] 30 sec. 72° C.

[0177] At the end of the program, the reaction vial is removed from thethermal cycler and the aqueous phase transferred to a new vial,extracted with phenol/chloroform (50:50 vol), and ethanol precipitated,and the DNA is recovered by standard procedures. This material issubsequently subjected to appropriate treatments for insertion into avector.

[0178] Cassette mutagenesis is another method useful for preparingvariants and is based on the technique described by Wells et al. (1985)Gene 34:315.

[0179] Additionally, the so-called phagemid display method may be usefulin making amino acid sequence variants of native or variant NRG3s ortheir fragments. This method involves 1) constructing a replicableexpression vector comprising a first gene encoding a receptor to bemutated, a second gene encoding at least a portion of a natural orwild-type phage coat protein wherein the first and second genes areheterologous, and a transcription regulatory element operably linked tothe first and second genes, thereby forming a gene fusion encoding afusion protein; 2) mutating the vector at one or more selected positionswithin the first gene thereby forming a family of related plasmids; 3)transforming suitable host cells with the plasmids; 4) infecting thetransformed host cells with a helper phage having a gene encoding thephage coat protein; 5) culturing the transformed infected host cellsunder conditions suitable for forming recombinant phagemid particlescontaining at least a portion of the plasmid and capable of transformingthe host, the conditions adjusted so that no more than a minor amount ofphagemid particles display more than one copy of the fusion protein onthe surface of the particle; 6) contacting the phagemid particles with asuitable antigen so that at least a portion of the phagemid particlesbind to the antigen; and 7) separating the phagemid particles that bindfrom those that do not. Steps 4 through 7 can be repeated one or moretimes. Preferably in this method the plasmid is under tight control ofthe transcription regulatory element, and the culturing conditions areadjusted so that the amount or number of phagemid particles displayingmore than one copy of the fusion protein on the surface of the particleis less than about 1%. Also, preferably, the amount of phagemidparticles displaying more than one copy of the fusion protein is lessthan 10% of the amount of phagemid particles displaying a single copy ofthe fusion protein. Most preferably, the amount is less than 20%.Typically in this method, the expression vector will further contain asecretory signal sequence fused to the DNA encoding each subunit of thepolypeptide and the transcription regulatory element will be a promotersystem. Preferred promoter systems are selected from lac Z, λ_(PL), tac,T7 polymerase, tryptophan, and alkaline phosphatase promoters andcombinations thereof. Also, normally the method will employ a helperphage selected from M13K07, M13R408, M13-VCS, and Phi X 174. Thepreferred helper phage is M13K07, and the preferred coat protein is theM13 Phage gene III coat protein. The preferred host is E. coli, andprotease-deficient strains of E. coli.

[0180] Further details of the foregoing and similar mutagenesistechniques are found in general textbooks, such as, for example,Sambrook et al., supra, and Current Protocols in Molecular Biology,Ausubel et al. eds., supra.

[0181] F. Glycosylation Variants.

[0182] Glycosylation variants are included within the scope of thepresent invention. They include variants completely lacking inglycosylation (unglycosylated), variants having at least one lessglycosylated site than the native form (deglycosylated) as well asvariants in which the gycosylation has been changed. Included aredeglycosylated and unglycosylated amino acid sequences variants,deglycosylated and unglycosylated native NRG3s or fragments thereof andother glycosylation variants. For example, substitutional or deletionalmutagenesis may be employed to eliminate the N- or O-linkedglycosylation sites in the a native or variant NRG3 of the presentinvention, e.g. the asparagine residue may be deleted or substituted foranother basic residue such as lysine or histidine. Alternatively,flanking residues making up the glycosylation site may be substituted ordeleted, even though the asparagine residues remain unchanged, in orderto prevent glycosylation by eliminating the glycosylation recognitionsite. Where the preferred NRL variant is the EGF-like domain of NRG3,the fragment is preferably unglycosylated.

[0183] Additionally, unglycosylated NRG3s which have the glycosylationsites of a native molecule may be produced in recombinant prokaryoticcell culture because prokaryotes are incapable of introducingglycosylation into polypeptides.

[0184] Glycosylation variants may be produced by appropriate host cellsor by in vitro methods. Yeast and insect cells, for example, introduceglycosylation which varies significantly from that of mammalian systems.Similarly, mammalian cells having a different species (e.g. hamster,murine, porcine, bovine or ovine), or tissue origin (e.g. lung, liver,lymphoid, mesenchymal or epidermal) than the source of the NRG3 areroutinely screened for the ability to introduce variant glycosylation ascharacterized for example by elevated levels of mannose or variantratios of mannose, fucose, sialic acid, and other sugars typically foundin mammalian glycoproteins. In vitro processing of the NRG3 typically isaccomplished by enzymatic hydrolysis, e.g. neuraminidate digestion.

[0185] G. Covalent Modifications.

[0186] Covalent modifications of the novel NRG3s of the presentinvention are included within the scope of the invention. Suchmodifications are traditionally introduced by reacting targeted aminoacid residues of the NRG3s with an organic derivatizing agent that iscapable of reacting with selected amino acid side chains or terminalresidues, or by harnessing mechanisms of post-translationalmodifications that function in selected recombinant host cells. Theresultant covalent derivatives are useful in programs directed atidentifying residues important for biological activity, for immunoassaysof the NRG3, or for the preparation of anti-NRG3 antibodies forimmunoaffinity purification of the recombinant. For example, completeinactivation of the biological activity of the protein after reactionwith ninhydrin would suggest that at least one arginyl or lysyl residueis critical for its activity, whereafter the individual residues whichwere modified under the conditions selected are identified by isolationof a peptide fragment containing the modified amino acid residue. Suchmodifications are within the ordinary skill in the art and are performedwithout undue experimentation.

[0187] Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the NRG3s with polypeptides as well as forcross-linking the NRG3 polypeptide to a water insoluble support matrixor surface for use in assays or affinity purification. In addition, astudy of interchain cross-links will provide direct information onconformational structure. Commonly used cross-linking agents include1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

[0188] Certain post-translational modifications are the result of theaction of recombinant host cells on the expressed polypeptide.Glutaminyl and aspariginyl residues are frequently post-translationallydeamidated to the corresponding glutamyl and aspartyl residues.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

[0189] Other post-translational modifications include hydroxylation ofproline and lysine, phosphorylation of hydroxyl groups of seryl,threonyl or tyrosyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton (1983)Proteins: Structure and Molecular Properties, W. H. Freeman & Co., SanFrancisco, pp. 79-86).

[0190] Further derivatives of the NRG3s herein are the so called“immunoadhesins”, which are chimeric antibody-like molecules combiningthe functional domain(s) of a binding protein (usually a receptor, acell-adhesion molecule or a ligand) with the an immunoglobulin sequence.The most common example of this type of fusion protein combines thehinge and Fc regions of an immunoglobulin (Ig) with domains of acell-surface receptor that recognizes a specific ligand. This type ofmolecule is called an “immunoadhesin”, because it combines “immune” and“adhesion” functions; other frequently used names are “Ig-chimera”,“Ig-” or “Fc-fusion protein”, or “receptor-globulin.”

[0191] Immunoadhesins reported in the literature include, for example,fusions of the T cell receptor (Gascoigne et al. (1987) Proc. Natl.Acad. Sci. USA 84:2936-2940); CD4 (Capon et al. (1989) Nature337:525-531; Traunecker et al. (1989) Nature 339:68-70; Zettmeissl etal. (1990) DNA Cell Biol. USA 9:347-353; Byrn et al. (1990) Nature344:667-670); L-seNRG3 (homing receptor) (Watson et al. (1990) J. Cell.Biol. 110:2221-2229); Watson et al. (1991) Nature 349:164-167); E-seNRG3(Mulligan et al (1993) J. Immunol. 151:6410-17; Jacob et al.(1995)Biochemistry 34:1210-1217); P-seNRG3 (Mulligan et al., supra;Hollenbaugh et al. (1995) Biochemistry 34:5678-84); ICAM-1 (Stauton etal. (1992) J. Exp. Med. 176:1471-1476; Martin et al. (1993) J. Virol.67:3561-68; Roep et al. (1994) Lancet 343:1590-93); ICAM-2 (Damle et al.(1992) J. Immunol. 148:665-71); ICAM-3 (Holness et al. (1995) J. Biol.Chem. 270:877-84); LFA-3 (Kanner et al. (1992) J. Immunol. 148:23-29);L1 glycoprotein (Doherty et al. (1995) Neuron 14:57-66); TNF-R1(Ashkenazi et al., (1991) Proc. Natl. Acad. Sci. USA 88:10535-539);Lesslauer et al. (1991) Eur. J. Immunol. 21:2883-86; Peppel et al (1991)J. Exp. Med. 174:1483-1489); TNF-R2 (Zack et al. (1993) Proc. Natl.Acad. Sci. USA 90:2335-39; Wooley et al (1993) J. Immunol. 151:6602-07);CD44 (Aruffo et al. (1990) Cell 61:1303-1313); CD28 and B7 (Linsley etal. (1991) J. Exp. Med. 173:721-730); CTLA-4 (Lisley et al. (1991) J.Exp. Med. 174:561-569); CD22 (Stamenkovic et al. (1991) Cell66:1133-1144); NP receptors (Bennett et al. (1991) J. Biol. Chem.266:23060-23067); IgE receptor α (Ridgway and Gorman (1991) J. Cell.Biol. 115:1448 abstr.); IFN-γ R α-and β-chain (Marsters et al. (1995)Proc. Natl. Acad. Sci. USA 92:5401-05); trk-A, -B, and -C (Shelton etal. (1995) J. Neurosci. 15:477-91); IL-2 (Landolfi (1991) J. Immunol.146:915-19); IL-10 (Zheng et al. (1995) J. Immunol. 154:5590-5600).

[0192] The simplest and most straightforward immunoadhesin designcombines the binding region(s) of the ‘adhesin’ protein with the hingeand Fc regions of an immunoglobulin heavy chain. Ordinarily, whenpreparing the NRG3-immunoglobulin chimeras of the present invention,nucleic acid encoding the desired NRG3 polypeptide will be fused at theC-terminus of the desired sequence to the N-terminus of a nucleic acidsequence encoding an immunoglobulin constant domain sequence, howeverfusion to the N-terminus of the desired NRG3 sequence is also possible.Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion or bindingcharacteristics of the NRG3-immunoglobulin chimeras.

[0193] In a preferred embodiment, the sequence of a native, mature NRG3polypeptide, or a soluble form thereof such as a (transmembranedomain-inactivated or EGF-like domain polypeptide) form thereof, isfused to the N-terminus of the C-terminal portion of an antibody (inparticular the Fc domain), containing the effector functions of animmunoglobulin, e.g. IgG-1. It is possible to fuse the entire heavychain constant region to the NRG3 sequence. However, more preferably, asequence beginning in the hinge region just upstream of the papaincleavage site (which defines IgG Fc chemically; residue 216, taking thefirst residue of heavy chain constant region to be 114 (Kobet et al.,supra), or analogous sites of other immunoglobulins) is used in thefusion. In a particularly preferred embodiment, the NRG3 sequence (fulllength or soluble) is fused to the hinge region and CH2 and CH3 or CH1,hinge, CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavy chain. Theprecise site at which the fusion is made is not critical, and theoptimal site can be determined by routine experimentation.

[0194] In some embodiments, the NRG3-immunoglobulin chimeras areassembled as multimers, and particularly as homo-dimers or -tetramers(WO 91/08298). Generally, these assembled immunoglobulins will haveknown unit structures. A basic four chain structural unit is the form inwhich IgG, IgD, and IgE exist. A four unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer ofbasic four units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each four unit may be the same or different.

[0195] Various exemplary assembled NRG3-immunoglobulin chimeras withinthe scope of the invention are schematically diagrammed below:

[0196] (a) ACL-AC_(L);

[0197] (b) AC_(H)-[AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H)];

[0198] (c) AC_(L)-AC_(H)-[AC_(L)-AC_(H), AC_(L)-V_(H)C_(H),V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H)];

[0199] (d) AC_(L)-V_(H)C_(H)-[AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H)];

[0200] (e) V_(L)C_(L)-AC_(H)-[AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)];and

[0201] (f) [A-Y]_(n)-[V_(L)C_(L)-V_(H)C_(H)]₂,

[0202] wherein

[0203] each A represents identical or different novel NRG3 polypeptideamino acid sequences;

[0204] V_(L) is an immunoglobulin light chain variable domain;

[0205] V_(H) is an immunoglobulin heavy chain variable domain;

[0206] C_(L) is an immunoglobulin light chain constant domain;

[0207] C_(H) is an immunoglobulin heavy chain constant domain;

[0208] n is an integer greater than 1;

[0209] Y designates the residue of a covalent cross-linking agent.

[0210] In the interest of brevity, the foregoing structures only showkey features; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

[0211] Although the presence of an immunoglobulin light chain is notrequired in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently associatedto an NRG3-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the NRG3 polypeptide. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the NRG3-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodssuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

[0212] In a preferred embodiment, the immunoglobulin sequences used inthe construction of the immunoadhesins of the present invention are froman IgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG-1 and IgG-3 immunoglobulinsequences is preferred. A major advantage of using IgG-1 is that IgG-1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG-3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG-3hinge is longer and more flexible, so it can accommodate larger‘adhesin’ domains that may not fold or function properly when fused toIgG-1. While IgG immunoadhesins are typically mono- or bivalent, otherIg subtypes like IgA and IgM may give rise to dimeric or pentamericstructures, respectively, of the basic Ig homodimer unit. Multimericimmunoadhesins are advantageous in that they can bind their respectivetargets with greater avidity than their IgG-based counterparts. Reportedexamples of such structures are CD4-IgM (Traunecker et al., supra);ICAM-IgM (Martin et al. (1993) J. Virol. 67:3561-68); and CD2-IgM(Arulanandam et al. (1993) J. Exp. Med. 177:1439-50).

[0213] For NRG3-Ig immunoadhesins, which are designed for in vivoapplication, the pharmacokinetic properties and the effector functionsspecified by the Fc region are important as well. Although IgG-1, IgG-2and IgG-4 all have in vivo half-lives of 21 days, their relativepotencies at activating the complement system are different. IgG-4 doesnot activate complement, and IgG-2 is significantly weaker at complementactivation than IgG-1. Moreover, unlike IgG-1, IgG-2 does not bind to Fcreceptors on mononuclear cells or neutrophils. While IgG-3 is optimalfor complement activation, its in vivo half-life is approximately onethird of the other IgG isotypes. Another important consideration forimmunoadhesins designed to be used as human therapeutics is the numberof allotypic variants of the particular isotype. In general, IgGisotypes with fewer serologically-defined allotypes are preferred. Forexample, IgG-1 has only four serologically-defined allotypic sites, twoof which (G1m and 2) are located in the Fc region; and one of thesesites G1m1, is non-immunogenic. In contrast, there are 12serologically-defined allotypes in IgG-3, all of which are in the Fcregion; only three of these sites (G3m5, 11 and 21) have one allotypewhich is nonimmunogenic. Thus, the potential immunogenicity of a γ3immunoadhesin is greater than that of a γ1 immunoadhesin.

[0214] NRG3-Ig immunoadhesins are most conveniently constructed byfusing the cDNA sequence encoding the NRG3 portion in-frame to an IgcDNA sequence. However, fusion to genomic Ig fragments can also be used(see, e.g. Gascoigne et al. (1987) Proc. Natl. Acad. Sci. USA84:2936-2940; Aruffo et al. (1990) Cell 61:1303-1313; Stamenkovic et al.(1991) Cell 66:1133-1144). The latter type of fusion requires thepresence of Ig regulatory sequences for expression. cDNAs encoding IgGheavy-chain constant regions can be isolated based on published sequencefrom cDNA libraries derived from spleen or peripheral blood lymphocytes,by hybridization or by polymerase chain reaction (PCR) techniques.

[0215] Other derivatives of the novel NRG3s of the present invention,which possess a longer half-life than the native molecules comprise theNRG3, NRG3 fragment (such as the EGF-like domain) or aNRG3-immunoglobulin chimera, covalently bonded to a nonproteinaceouspolymer. The nonproteinaceous polymer ordinarily is a hydrophilicsynthetic polymer, i.e., a polymer not otherwise found in nature.However, polymers which exist in nature and are produced by recombinantor in vitro methods are useful, as are polymers which are isolated fromnative sources. Hydrophilic polyvinyl polymers fall within the scope ofthis invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.Particularly useful are polyalkylene ethers such as polyethylene glycol(PEG); polyelkylenes such as polyoxyethylene, polyoxypropylene, andblock copolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextranesulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparon. Thepolymer prior to cross-linking need not be, but preferably is, watersoluble, but the final conjugate must be water soluble. In addition, thepolymer should not be highly immunogenic in the conjugate form, norshould it possess viscosity that is incompatible with intravenousinfusion or injection if it is intended to be administered by suchroutes.

[0216] Preferably the polymer contains only a single group which isreactive. This helps to avoid cross-linking of protein molecules.However, it is within the scope herein to optimize reaction conditionsto reduce cross-linking, or to purify the reaction products through gelfiltration or chromatographic sieves to recover substantially homogenousderivatives.

[0217] The molecular weight of the polymer may desirably range fromabout 100 to 500,000, and preferably is from about 1,000 to 20,000. Themolecular weight chosen will depend upon the nature of the polymer andthe degree of substitution. In general, the greater the hydrophilicityof the polymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

[0218] The polymer generally is covalently linked to the novel NRG3,NRG3 fragment or to the NRG3-immunoglobulin chimeras through amultifunctional crosslinking agent which reacts with the polymer and oneor more amino acid or sugar residues of the NRG3 or NRG3-immunoglobulinchimera to be linked. However, it is within the scope of the inventionto directly crosslink the polymer by reacting a derivatized polymer withthe hybrid, or vice versa.

[0219] The covalent crosslinking site on the NRG3 or NRG3-Ig includesthe N-terminal amino group and epsilon amino groups found on lysineresidues, as well as other amino, imino, carboxyl, sulfhydryl, hydroxylor other hydrophilic groups. The polymer may be covalently bondeddirectly to the hybrid without the use of a multifunctional (ordinarilybifunctional) crosslinking agent. Covalent binding to amino groups isaccomplished by known chemistries based upon cyanuric chloride, carbonyldiimidazole, aldehyde reactive groups (PEG alkoxide plus diethyl acetalof bromoacetaldehyde; PEG plus DMSO and acetic anhydride, or PEGchloride plus the phenoxide of 4-hydroxybenzaldehyde, succinimidylactive esters, activated dithiocarbonate PEG,2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activatedPEG.) Carboxyl groups are derivatized by coupling PEG-amine usingcarbodiimide.

[0220] Polymers are conjugated to oligosaccharide groups by oxidationusing chemicals, e.g. metaperiodate, or enzymes, e.g. glucose orgalactose oxidase, (either of which produces the aldehyde derivative ofthe carbohydrate), followed by reaction with hydrazide or aminoderivatized polymers, in the same fashion as is described by Heitzmannet al. (1974) P.N.A.S. 71:3537-41 or Bayer et al. (1979) Methods inEnzymology 62:310, for the labeling of oligosaccharides with biotin oravidin. Further, other chemical or enzymatic methods which have beenused heretofore to link oligosaccharides are particularly advantageousbecause, in general, there are fewer substitutions than amino acid sitesfor derivatization, and the oligosaccharide products thus will be morehomogenous. The oligosaccharide substituents also are optionallymodified by enzyme digestion to remove sugars, e.g. by neuraminidasedigestion, prior to polymer derivatization.

[0221] The polymer will bear a group which is directly reactive with anamino acid side chain, or the N- or C-terminus of the polypeptidelinked, or which is reactive with the multifunctional cross-linkingagent. In general, polymers bearing such reactive groups are known forthe preparation of immobilized proteins. In order to use suchchemistries here, one should employ a water soluble polymer otherwisederivatized in the same fashion as insoluble polymers heretoforeemployed for protein immobilization. Cyanogen bromide activation is aparticularly useful procedure to employ in crosslinking polysaccharides.

[0222] “Water soluble” in reference to the starting polymer means thatthe polymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.“Water soluble” in reference to the polymer conjugate means that theconjugate is soluble in physiological fluids such as blood.

[0223] The degree of substitution with such a polymer will varydepending upon the number of reactive sites on the protein, whether allor a fragment of the protein is used, whether the protein is a fusionwith a heterologous protein (e.g. a NRG3-immunoglobulin chimera), themolecular weight, hydrophilicity and other characteristics of thepolymer, and the particular protein derivatization sites chosen. Ingeneral, the conjugate contains about from 1 to 10 polymer molecules,while any heterologous sequence may be substituted with an essentiallyunlimited number of polymer molecules so long as the desired activity isnot significantly adversely affected. The optimal degree ofcross-linking is easily determined by an experimental matrix in whichthe time, temperature and other reaction conditions are varied to changethe degree of substitution, after which the ability of the conjugates tofunction in the desired fashion is determined.

[0224] The polymer, e.g. PEG, is cross-linked by a wide variety ofmethods known per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG. Certain of these methods,however, are not preferred for the purposes herein. Cyanuronic chloridechemistry leads to many side reactions, including protein cross-linking.In addition, it may be particularly likely to lead to inactivation ofproteins containing sulfhydryl groups. Carbonyl diimidazole chemistry(Beauchamp et al. (1983) Anal Biochem. 131:25-33) requires high pH(>8.5), which can inactivate proteins. Moreover, since the “activatedPEG” intermediate can react with water, a very large molar excess of“activated PEG” over protein is required. The high concentrations of PEGrequired for the carbonyl diimidazole chemistry also led to problems inpurification, as both gel filtration chromatography and hydrophilicinteraction chromatography are adversely affected. In addition, the highconcentrations of “activated PEG” may precipitate protein, a problemthat per se has been noted previously (Davis, U.S. Pat. No. 4,179,337).On the other hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531)is more efficient since it requires only a 40-fold molar excess of PEGand a 1-2 hr incubation. However, the manganese dioxide suggested byRoyer for preparation of the PEG aldehyde is problematic “because of thepronounced tendency of PEG to form complexes with metal-based oxidizingagents” (Harris et al. (1984) J. Polym. Sci. Polym. Chem. Ed.22:341-52). The use of a Moffatt oxidation, utilizing DMSO and aceticanhydride, obviates this problem. In addition, the sodium borohydridesuggested by Royer must be used at high pH and has a significanttendency to reduce disulfide bonds. In contrast, sodiumcyanoborohydride, which is effective at neutral pH and has very littletendency to reduce disulfide bonds is preferred.

[0225] The long half-life conjugates of this invention are separatedfrom the unreacted starting materials by gel filtration. Heterologousspecies of the conjugates are purified from one another in the samefashion. The polymer also may be water-insoluble, as a hydrophilic gel.

[0226] The novel NRG3s may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

[0227] H. Antibody Preparation.

[0228] (i) Polyclonal Antibodies

[0229] Polyclonal antibodies to a NRG3, or fragment thereof (such as theEGF-like domain) of the present invention generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the NRG3 and an adjuvant. It may be useful to conjugate the NRG3 or afragment containing the target amino acid sequence to a protein that isimmunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹are different alkyl groups.

[0230] Animals are immunized against the immunogenic conjugates orderivatives by combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with ⅕ to {fraction (1/10)} the original amount ofconjugate in Freud's complete adjuvant by subcutaneous injection atmultiple sites. 7 to 14 days later the animals are bled and the serum isassayed for anti-NRG3 antibody titer. Animals are boosted until thetiter plateaus. Preferably, the animal boosted with the conjugate of thesame NRG3, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

[0231] (ii) Monoclonal Antibodies

[0232] Monoclonal antibodies are obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Thus, the modifier “monoclonal” indicates the character of the antibodyas not being a mixture of discrete antibodies. For example, theanti-NRG3 monoclonal antibodies of the invention may be made using thehybridoma method first described by Kohler and Milstein (1975) Nature256:495, or may be made by recombinant DNA methods (Cabilly, et al.,U.S. Pat. No. 4,816,567).

[0233] DNA encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells of the invention serve as a preferred source of suchDNA. Once isolated, the DNA may be placed into expression vectors, whichare then transfected into host cells such as simian COS cells, Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The DNA also may be modified,for example, by substituting the coding sequence for human heavy andlight chain constant domains in place of the homologous murinesequences, Morrison, et al. (1984) Proc. Nat. Acad. Sci. 81:6851, or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of a NRG3 monoclonal antibody herein.

[0234] Typically such non-immunoglobulin polypeptides are substitutedfor the constant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a NRG3 andanother antigen-combining site having specificity for a differentantigen.

[0235] Chimeric or hybrid antibodies also may be prepared in vitro usingknown methods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

[0236] For diagnostic applications, the antibodies of the inventiontypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, 32P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

[0237] Any method known in the art for separately conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter, et al. (1962) Nature 144:945; David, et al.(1974) Biochemistry 13:1014; Pain, et al. (1981) J. Immunol. Meth.40:219; and Nygren (1982) J. Histochem. and Cytochem. 30:407.

[0238] The antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc., 1987).

[0239] (iii) Humanized Antibodies

[0240] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0241] It is important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e. theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.For further details see PCT/US93/07832, which is a continuation-in-partof PCT/US92/05126, which references are herein incorporated by referencein their entirety.

[0242] Alternatively, it is now possible to produce transgenic animals(e.g. mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA90:2551-255; Jakobovits et al. (1993) Nature 362:255-258.

[0243] (iv) Bispecific Antibodies

[0244] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for a NRG3 of the present invention the other one isfor any other antigen, for example, another member of the NRG3 family.Such constructs can also be referred to as bispecific immunoadhesins.

[0245] Traditionally, the recombinant production of bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two heavy chains have differentspecificities (Millstein and Cuello (1983) Nature 305:537-539). Becauseof the random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al. (1991) EMBO 10:3655-3659. This problem may be overcomeby selecting a common light chain for each arm o the bispecific antibodysuch that binding specificity of each antibody is maintained, asdisclosed in U.S. application Ser. No. 08/850058, filed May 5, 1997.

[0246] According to a different and more preferred approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, and secondand third constant regions of an immunoglobulin heavy chain (CH2 andCH3). It is preferred to have the first heavy chain constant region(CHI) containing the site necessary for light chain binding, present inat least one of the fusions. DNAs encoding the immunoglobulin heavychain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inPCT application WO 94/04690 published Mar. 3, 1994.

[0247] For further details of generating bispecific antibodies see, forexample, Suresh et al. (1986) Methods in Enzymology 121:210.

[0248] (v) Heteroconjugate Antibodies

[0249] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

[0250] I. Diagnostic Kits and Articles of Manufacture.

[0251] Since the invention provides a diagnostic assay (i.e. fordetecting neurological disorders and for detecting the presence of NRG3in a sample using antibodies or DNA markers) as a matter of convenience,the reagents for these assays can be provided in a kit, i.e., a packagedcombination of reagents, for combination with the sample to be tested.The components of the kit will normally be provided in predeterminedratios. Thus, a kit may comprise the antibody or NRG3 (DNA orpolypeptide or fragment thereof) labeled directly or indirectly with asuitable label. Where the detectable label is an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. Particularly, the reagents may be provided as dry powders,usually lyophilized, including excipients which on dissolution willprovide a reagent solution having the appropriate concentration. The kitalso suitably includes instructions for carrying out the bioassay.

[0252] In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the neurologicaldisorders described herein is provided. The article of manufacturecomprises a container and a label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for treating thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The active agent in the compositionis NRG3 or an agonist or antagonist thereof. The label on, or associatedwith, the container indicates that the composition is used for treatingthe condition of choice. The article of manufacture may further comprisea second container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

[0253] J. Peptide and Non-peptide Analogs.

[0254] Peptide analogs of the NRG3s of the present invention are modeledbased upon the three-dimensional structure of the native polypeptides.Peptides may be synthesized by well known techniques such as thesolid-phase synthetic techniques initially described in Merrifield(1963) J. Am. Chem. Soc. 15:2149-2154. Other peptide synthesistechniques are, for examples, described in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2nd Ed., 1976, as well as in otherreference books readily available for those skilled in the art. Asummary of peptide synthesis techniques may be found in Stuart andYoung, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford,Ill. (1984). Peptides may also be prepared by recombinant DNAtechnology, using a DNA sequence encoding the desired peptide.

[0255] In addition to peptide analogs, the present invention alsocontemplates non-peptide (e.g. organic) compounds which displaysubstantially the same surface as the peptide analogs of the presentinvention, and therefore interact with other molecules in a similarfashion.

[0256] K. Uses of the NRG3s.

[0257] Amino acid sequence variants of the native NRG3s of the presentinvention may be employed therapeutically to compete with the normalbinding of the native proteins to their receptor, ErbB4. The NRG3 aminoacid sequence variants are, therefore, useful as competitive inhibitorsof the biological activity of native NRG3s.

[0258] Native NRG3s and their amino acid sequence variants are useful inthe identification and purification of the native ErbB4 receptor. Thepurification is preferably performed by immunoadhesins comprising a NRG3amino acid sequence retaining the qualitative ability of a native NRG3of the present invention to recognize its native ErbB4 receptor.

[0259] The native NRG3s of the present invention are further useful asmolecular markers of the tissues in which the ErbB4 receptor isexpressed.

[0260] Furthermore, the NRG3s, preferably the EGF-like domain of theNRG3 of the present invention, provide valuable sequence motifs whichcan be inserted or substituted into other native members of the NRG3family of molecules, such as the heregulins. The alteration of thesenative proteins by the substitution or insertion of sequences from thenovel NRG3s of the present invention can yield variant molecules withaltered biological properties, such as receptor binding affinity orreceptor specificity. For example, one or more NRG3 domains of anothermember of the NRG3 family may be entirely or partially replaced by NRG3domain sequences derived from the NRG3s of the present invention.Similarly, EGF-like domain sequences from the NRG3s herein may besubstituted or inserted into the amino acid sequences of other NRG3s.

[0261] Nucleic acid encoding the NRG3s of the present invention is alsouseful in providing hybridization probes for searching cDNA and genomiclibraries for the coding sequence of other NRG3s.

[0262] Additionally, NRG3s of the invention are useful in kits for thediagnosis of disease related to NRG3 and for methods of detecting thepresence or absence of NRG3 in a sample, such as a body fluid, asdescribed herein.

[0263] Binding and activation of the ErbB4 receptor by NRG3 is expectedto mediate such physiological responses in cells expressing the ErbB4receptor as cell growth, cell proliferation, and cell differentiationparticularly in neural tissue. As a result, mammalian NRG3, or an ErbB4receptor binding and activating fragment thereof, is useful in thetreatment of diseases in which neural cell growth, proliferation and/ordifferentiation alleviate symptoms of the disease. The NRG3 may be thefull length amino acid sequence of the murine NRG3 (SEQ ID NO: 2) or thehuman NRG3s (SEQ ID NO: 6 or SEQ ID NO: 23); the full length amino acidsequence from another mammalian species having at least approximately75% homology to the murine and human NRG3 at the amino acid level,preferably about 90% amino acid sequence homology in the EGF-likebinding domain; and an amino acid sequence comprising the EGF-likedomain of NRG3, which sequence binds to the ErbB4 receptor. Where theNRG3 or ErbB4 receptor binding fragment is agonist, the NRG3 or fragmentbinds to and activates ErbB4 receptor. Where the NRG3 or fragment is anantagonist, the NRG3 or fragment binds to but does not activate ErbB4receptor, thereby preventing activation by the naturally occurring NRG3or agonist.

[0264] Diseases treatable by administration of NRG3 or an agonistthereof (such as a polypeptide comprising an NRG3 EGF-like domain)include, but are not limited to, disorders that may arise in a patientin whom the nervous system has been damaged by, e.g., trauma, surgery,stroke, ischemia, infection, metabolic disease, nutritional deficiency,malignancy, or toxic agents; motoneuron disorders, such as amyotrophiclateral sclerosis (Lou Gehrig's disease), Bell's palsy, and variousconditions involving spinal muscular atrophy, or paralysis; human“neurodegenerative disorders”, such as Alzheimer's disease, Parkinson'sdisease, epilepsy, multiple sclerosis, Huntington's chorea, Down'sSyndrome, nerve deafness, and Meniere's disease; neuropathy, andespecially peripheral, referring to a disorder affecting the peripheralnervous system, most often manifested as one or a combination of motor,sensory, sensorimotor, or autonomic neural dysfunction, such as distalsensorimotor neuropathy, or autonomic neuropathies including reducedmotility of the gastrointestinal tract or atony of the urinary bladder.Examples of neuropathies associated with systemic disease includepost-polio syndrome; examples of hereditary neuropathies includeCharcot-Marie-Tooth disease, Refsum's disease, Abetalipoproteinemia,Tangier disease, Krabbe's disease, Metachromatic leukodystrophy, Fabry'sdisease, and Dejerine-Sottas syndrome; and examples of neuropathiescaused by a toxic agent include those caused by treatment with achemotherapeutic agent such as vincristine, cisplatin, methotrexate, or3′-azido-3′-deoxythymidine. Also, NRG3 or biologically active fragmentsthereof (such as an EGF-like domain of an NRG3) may be used to treatdiseases of skeletal muscle of smooth muscle, such as muscular dystrophyor diseases caused by skeletal or smooth muscle wasting.

[0265] Semipermeable, implantable membrane devices are useful as meansfor delivering drugs in certain circumstances. For example, cells thatsecrete soluble NRG3, or agonist thereof, or chimeras can beencapsulated, and such devices can be implanted into a patient, forexample, into the brain of patients suffering from Parkinson's Disease.See, U.S. Pat. No. 4,892,538 of Aebischer et al.; U.S. Pat. No.5,011,472 of Aebischer et al.; U.S. Pat. No. 5,106,627 of Aebischer etal.; PCT Application WO 91/10425; PCT Application WO 91/10470; Winn etal. (1991) Exper. Neurology 113: 322-329; Aebischer et al. (1991) Exper.Neurology 111:269-275; and Tresco et al. (1992) ASAIO 38:17-23.Accordingly, also included is a method for preventing or treating damageto a nerve or damage to other NRG3-expressing or NRG3-responsive cells,e.g. brain, heart, or kidney cells, as taught herein, which methodcomprises implanting cells that secrete NRG3, or fragment or agonistthereof, or antagonist as may be required for the particular condition,into the body of patients in need thereof. Finally, the presentinvention includes an implantation device, for preventing or treatingnerve damage or damage to other cells as taught herein, containing asemipermeable membrane and a cell that secretes NRG3, or fragment oragonist thereof, (or antagonist as may be required for the particularcondition) encapsulated within the membrane, the membrane beingpermeable to NRG3, or fragment agonist thereof, and impermeable tofactors from the patient detrimental to the cells. The patient's owncells, transformed to produce NRG3 ex vivo, could be implanted directlyinto the patient, optionally without such encapsulation. The methodologyfor the membrane encapsulation of living cells is familiar to those ofordinary skill in the art, and the preparation of the encapsulated cellsand their implantation in patients may be accomplished readily as isknown in the art. The present invention includes, therefore, a methodfor preventing or treating cell damage, preferably nerve damage, byimplanting cells into the body of a patient in need thereof, the cellseither selected for their natural ability to generate NRG3, or fragmentor agonist thereof, or engineered to secrete NRG3, or fragment oragonist thereof. Preferably, the secreted NRG3 is soluble, human NRG3when the patient is human. The implants are preferably non-immunogenicand/or prevent immunogenic implanted cells from being recognized by theimmune system. For CNS delivery, a preferred location for the implant isthe cerebral spinal fluid of the spinal cord.

[0266] The administration of the NRG3, fragment or variant thereof, ofthe present invention can be done in a variety of ways, e.g., thoseroutes known for specific indications, including, but not limited to,orally, subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraarterially, intralesionally,intraventricularly in the brain, or intraocularly. The NRG3 may beadministered continuously by infusion into the fluid reservoirs of theCNS, although bolus injection is acceptable, using techniques well knownin the art, such as pumps or implantation. Sustained release systems canbe used. Where the disorder permits, one may formulate and dose the NRG3variant for site-specific delivery. Administration can be continuous orperiodic. Administration can be accomplished by a constant- orprogrammable-flow implantable pump or by periodic injections.

[0267] Semipermeable, implantable membrane devices are useful as meansfor delivering drugs in certain circumstances. For example, cells thatsecrete soluble NGF variant can be encapsulated, and such devices can beimplanted into a patient, for example, into the brain or spinal chord(CSF) of a patient suffering from Parkinson's Disease. See, U.S. Pat.No. 4,892,538 of Aebischer et al.; U.S. Pat. No. 5,011,472 of Aebischeret al.; U.S. Pat. No. 5,106,627 of Aebischer et al.; PCT Application WO91/10425; PCT Application WO 91/10470; Winn et al. (1991) Exper.Neurology 113:322-329; Aebischer et al. (1991) Exper. Neurology111:269-275; and Tresco et al. (1992) ASAIO 38:17-23. Finally, thepresent invention includes an implantation device, for preventing ortreating nerve damage or damage to other cells as taught herein,containing a semipermeable membrane and a cell that secretes an NRG3,the cell being encapsulated within the membrane, and the membrane beingpermeable to NRG3, but impermeable to factors from the patientdetrimental to the cells. The patient's own cells, transformed toproduce NRG3 ex vivo, optionally could be implanted directly into thepatient without such encapsulation. The methodology for the membraneencapsulation of living cells is familiar to those of ordinary skill inthe art, and the preparation of the encapsulated cells and theirimplantation in patients may be accomplished readily as is known in theart. Preferably, the secreted NRG3, fragment or variant thereof, is ahuman NRG3 when the patient is human. The implants are preferablynon-immunogenic and/or prevent immunogenic implanted cells from beingrecognized by the immune system. For CNS delivery, a preferred locationfor the implant is the cerebral spinal fluid of the spinal cord.

[0268] The pharmaceutical compositions of the present invention comprisea NRG3 in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, and may include such physiologically acceptable materialsas carriers, excipients, stabilizers, buffers, salts, antioxidants,hydrophilic polymers, amino acids, carbohydrates, ionic or nonionicsurfactants, and polyethylene or propylene glycol. The NRG3 may be in atime-release form for implantation, or may be entrapped in microcapsulesusing techniques well known in the art.

[0269] An effective amount of NRG3 or NRG3 agonist or antagonist to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 10 ng/kg to up to 100 mg/kg of patient body weight or more perday, preferably about 1 μg/kg/day to 10 mg/kg/day. Typically, theclinician will administer NRG3 or NRG3 agonist or antagonist until adosage is reached that achieves the desired effect for treatment of theabove mentioned disorders.

[0270] L. Transgenic and Knockout Animals

[0271] Nucleic acids which encode novel NRG3 from non-human species,such as the murine NRG3, can be used to generate either transgenicanimals or “knock out” animals which, in turn, are useful in thedevelopment and screening of therapeutically useful reagents. Atransgenic animal (e.g., a mouse) is an animal having cells that containa transgene, which transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops. In one embodiment, murine cDNAencoding NRG3 or an appropriate sequence thereof can be used to clonegenomic DNA encoding NRG3 in accordance with established techniques andthe genomic sequences used to generate transgenic animals that containcells which express DNA encoding NRG3. Methods for generating transgenicanimals, particularly animals such as mice, have become conventional inthe art and are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009. Typically, particular cells, such as neuronal cells, would betargeted for NRG3 transgene incorporation with tissue-specificenhancers, which could result in altered cell differentiation, cellproliferation, or cellular apoptosis, depending upon the ligandinteraction with the expressed polypeptide. Transgenic animals thatinclude a copy of a transgene encoding NRG3 introduced into the germline of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding NRG3. Such animals can beused as tester animals for reagents thought to confer protection from,for example, diseases associated with abnormal neuronal differentiationand neuronal cell proliferation, for example. In accordance with thisfacet of the invention, an animal is treated with the reagent and areduced incidence of the disease, compared to untreated animals bearingthe transgene, would indicate a potential therapeutic intervention forthe disease.

[0272] Alternatively, the non-human homologues of NRG3 can be used toconstruct a NRG3 “knock out” animal which has a defective or alteredgene encoding NRG3 as a result of homologous recombination between theendogenous gene encoding NRG3 and altered genomic DNA encoding NRG3introduced into an embryonic cell of the animal. For example, murinecDNA encoding NRG3 can be used to clone genomic DNA encoding NRG3 inaccordance with established techniques. A portion of the genomic DNAencoding NRG3 can be deleted or replaced with another gene, such as agene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see e.g.,Thomas and Capecchi, Cell 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell 69: 915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can beused in the selection of potential therapeutic agents, such as NRG3agonists, that restore the cellular processes initiated or maintained bynative NRG3; or the knockout animals can be used in the study of theeffects of NRG3 mutations.

[0273] The instant invention is shown and described herein in what isconsidered to be the most practical, and the preferred embodiments. Itis recognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

EXAMPLES

[0274] The following examples are provided so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make the compounds and compositions of the invention and how topractice the methods of the invention and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to insure accuracy with respect to numbers used (e.g. amounts,temperature, etc.), but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in degrees C, and pressure is at or near atmospheric.

Example 1 Molecular Cloning of a Mouse and Human Novel NRG3

[0275] Novel NRG3 cDNAs were identified using an expressed sequence tagshown below: AATTTCTGCCGAAAACTGATTCCATCTTATCGGATCCAACAGACCACTTGGGGATTGAATTCATGGAGAGTGAAGAAGTTTATCAAAGGCAGGTGCTGTCAATTTCATGTATCATCTTTGGAATTGTCATCGTGGGCATGTTCTGTGCAGCATTCTACTTCAAAAGCAAGAAACAAGCTAAACAAATCCAAGAGCAGCTGAAAGTGCCACAAAATGGTAAAAGCTACAGTCTCAAAGCATCCAGCACAATGGCAAAGTCAGAGAACTTGGTGAAGAGCCATGTCCAGCTGCAAAATAAAATGTCAGGCTTCTGAGCCCAAGCTAAGCCATCATATCCCCTGTNGACCTGCACGTGCACATCCNGATGGCCCGTTTCCTGCCTTTTNTGATGACATTTNCACCACAAATGNAGTGAAAATGGGNCTTTTCNTGCCTTAACTGGTTGACNTTTTTNCCCCAAAAGGAG (EST; SEQ ID NO: 21; Genbank entry H2365 1) from theNational Center for Biotechnology Information (NCBI) database of ESTs.This EST from a human brain cDNA library, encodes an amino acid sequencehaving approximately 62% identity to amino acids 232-316 of heregulin-β1(also designated neuregulin-β1, or NRG1).

[0276] To obtain a partial human cDNA clone, a 50-base single strandedoligonucleotide probe(5′-TGGTAAAAGCTACAGTCTCAAAGCATCCAGCACAATGGCAAAGTCAGAGA-3′; SEQ ID NO:18) was synthesized based on the EST sequence. The probe was used toscreen 1.5 ×10⁶ plaques from a λgt10 cDNA library prepared from humanfetal brain RNA (HL3003a, Clontech) as described by Godowski et al.(Godowski, P. J. et al. (1989) PNAS USA 86:8083-8087, hereinincorporated by reference in its entirety). Nine positive plaques wereobtained and the sequences of both strands of the largest inserts weredetermined by standard sequencing techniques. From these clonedoverlapping sequences, a partial cDNA sequence of the human NRG3 wasobtained.

[0277] Additional 5′ human NRG3 sequence was obtained by anchored PCR ofhuman hippocampus RNA (Clontech). The complete human open reading framenucleic acid sequence deduced from direct sequencing of hNRG3B1 cDNA isshown in FIG. 2 (SEQ ID NO: 5). ATCC 209157 is nucleic acid comprisingan expression vector and the nucleotide sequence of the human NRG3B 1open reading frame. An alternatively spliced form of human NRG3 wascloned as pRK5.tk.neo.hNRG3B2 (SEQ ID NO: 22) encoding the deduced aminoacid sequence of SEQ ID NO: 23, which amino acid sequence lacks aminoacids 529 to 552 of SEQ ID NO: 6 (see FIG. 4B). Since this alternativelyspliced form of human NRG3 comprises the EGF-like domain of the otherNRG3s as well as high amino acid sequence homology, it is expected toexhibit the biological properties of the NRG3s disclosed herein.

[0278] To clone murine NRG3 cDNA sequences, two degenerate primers weredesigned based on regions proximal to the transmembrane domain of thepartial human cDNA, encoding the amino acid sequences NDGECFVI (SEQ IDNO: 19) and EFMESEEVY (SEQ ID NO: 20). A mouse brain cDNA library(Clontech, ML 1042a) was screened, and a clone (C5a) containing apartial murine NRG3 cDNA was obtained by standard techniques. Using aprobe derived from the C5a sequence, two additional mouse brain cDNAlibraries (ML1034h, Clontech; and 936309, Stratagene) were screened.Both strands of two overlapping murine partial NRG3 clones, SWAJ-3 andZAP-1 were sequenced and, together were found to encode an entire openreading frame (ORF) of 2139 bp having the DNA sequence SEQ ID NO: 1 andthe deduced amino acid sequence SEQ ID NO: 2 shown in FIG. 4A. Nucleicacid comprising the murine NRG3 open reading frame cloned into anexpression vector is designated pLXSN.mNRG3 (ATCC 209156).

[0279] The chromosomal localization of human NRG3 was mapped to 10q22 byPCR analysis of somatic cell hybrid DNA, whereas the NRG1 gene islocated at 8p11-22 (Lee, J. and Wood, W. I. (1993) Genomics 16: 790-791;and Orr-Urtreger, A. et al. (1993) Proc. Natl. Acad. Sci. USA90:1867-1871). Thus, NRG3 is a novel member of the EGF-like family ofprotein ligands.

Example 2 Characterization of the Mouse and Human NRG3 Deduced AminoAcid Sequences

[0280] The cDNAs of human and murine NRG3 contained open reading framesencoding proteins of 720 and 713 amino acids respectively, withpredicted MW of 77,901 Da for human NRG3 and 77,370 Da for murine NRG3(FIG. 4). The two species of NRG3 are 93% identical in amino acidsequence.

[0281] Analysis of the amino acid sequence of human NRG3 revealed thatit contained homology to NRG1 family members (i.e. 23% and 19% sequenceidentity to SMDF (Ho, W. H. et al. (1995) J. Biol. Chem. 270:14523-32)and heregulin-β1 (Holmes, W. E. et al. (1992) Science 256:1205-10)respectively). A hydropathy analysis indicated two hydrophobic segments:W⁶⁶-V⁹¹ and L³⁶²-F³⁸³ (amino acid numbers according to human NRG3).Similar to NRG1, the C-terminal hydrophobic segment may serve as thetransmembrane domain and the N-terminal region may act as internalsignal sequence (Wickner, W. T. and Lodish, H. F. (1985) Science230:400-7; Sabatini, D. D. et al. (1982) J. Cell Biol. 92:1-22; andBlobel, G. (1980) Proc. Natl. Acad. Sci. USA 77:1496-500). In contrastto many neuregulin family members, the extracellular domain of NRG3 isdevoid of Ig-like or kringle domains. Instead, NRG3 contains a uniqueAla/Gly rich segment at the N-terminus, a mucin-like Ser/Thr rich regioncontaining abundant sites for O-linked glycosylation, and an EGF motif.There are no predicted sites for N-linked glycosylation. The EGF-likedomain of NRG3 is distinct from those encoded by the NRG1 (31% identitycompared with neuregulin-β1 EGF-like domain) and NRG2 (39% identity withneuregulin- β1 EGF-like domain), suggesting that NRG3 is not analternatively spliced NRG1 isoform. A diagrammatic comparison ofEGF-like domains of EGF family members is shown in FIG. 5. The putativeintracellular domain of NRG3 contains only approximately 13% sequenceidentity to the intracellular domain of NRG1. The EGF-like domains ofthe EGF family members were obtained from the following sources, eachreference herein incorporated by reference in its entirety. Thesequences compared in FIG. 5 include the EGF-like domain of human NRG3(hNRG3.egf; SEQ ID NO: 4; disclosed herein); chicken ARIA (cARIA.egf;SEQ ID NO: 9) (Falls, D. L. et al. (1993) Cell 72:801-815), humanamphiregulin (hAR.egf; SEQ ID NO: 10) (Plowman, G. D. et al. (1990) Mol.Cell. Biol. 10:1969-81.); human betacellulin (hBTC.egf; SEQ ID NO: 11)(Sasada, R. et al. (1993) Biochem. Biophy. Res. Com. 190:1173-9); humanEGF (hEGF.egf; SEQ ID NO: 12)(Nagai, M. et al. (1985) Gene 36:183-8.);human heparin-binding EGF-like growth factor (hHB-EGF.egf; SEQ ID NO:13) (Higashiyama, S. et al. (1991) Science 251:936-9.); humanheregulin-α (hHRGα,; SEQ ID NO: 14); human heregulin-β (hHRGβ.egf, SEQID NO: 15)(Holmes, W. E. et al. (1992) Science 256:1205-1210); humanTGF-α (hTGFα.egf; SEQ ID NO: 16) (Derynck, R. et al. (1984) Cell38:287-97.); and mouse epiregulin (mEPR.egf; SEQ ID NO: 17) (Toyoda, H.et al. (1995) FEBS Lett. 377:403-7.).

Example 3 Expression of Murine and Human NRG3

[0282] A. Northern Blot Analysis of Human Tissue.

[0283] The tissue expression profile of the human NRG3 was examined byNorthern blot analysis. A multi-tissue RNA blot containing 2 μg each ofpoly(A)⁺RNA from human tissues were purchased from Clontech. The regionof the human NRG3 nucleic acid sequence encoding amino acids 394 to 536was used to generate DNA hybridization probes by PCR amplification. TheDNA probes were labeled with α-³²P-dCTP by random priming (Promega). TheRNA blot was hybridized with 50% formamide, 5× SSC, 50 mM potassiumphosphate (pH 7.0), 5× Denhardt's, 10% dextran sulfate at 42° C. for 20hr. The blot was washed with 0.1× SSC, 0.1%SDS at 50° C. for 30 min andexposed in Phospholmager™. Expression of NRG3 is mixtures of tissues wasused as a guide to determine expression in specific tissues by in situhybridization.

[0284] B. In Situ Hybridization Analysis of Mouse Tissues.

[0285] Formalin-fixed, paraffin-embedded mouse embryos (embryonic days13, 14, 16), and glutaraldehyde-fixed, paraffin-embedded orparaformaldehyde-fixed, frozen adult mouse brain, ovary, jejunum,kidney, adrenal, lung, stomach, spleen, skeletal muscle, liver and colonwere sectioned and processed for in situ hybridization by the method ofLu and Gillett (Lu, L. H. and Gillett, N. A. (1994) Cell Vision1:169-176) with modifications. Briefly, the in situ hybridization probewas generated by in vitro transcription directly from a PCR fragment,rather than from a plasmid DNA as described. ³²P-UTP-labeled sense andantisense riboprobes were generated by labeling PCR products of a cDNAfragment encoding amino acids C²⁹² to N⁴⁸² of murine NRG3.

[0286] C. Northern Blot And In Situ Hybridization Analyses Reveal aNeural Expression Pattern of NRG3.

[0287] A 4.4 kb mRNA transcript that hybridized to the probe derivedfrom amino acids 394 to 536 of human NRG3 was highly expressed in brain.In a Northern blot of various brain tissues, NRG3 expression wasdetected at high levels in most regions of the brain with the exceptionof corpus callosum. A lower level expression of a 1.9-kb transcript wasdetected in testis. The 4.4-kb transcript, but not the 1.9-kbtranscript, is of sufficient size to encode NRG3, suggesting that thesmaller transcript may encode an alternatively spliced form of NRG3. Asimilar pattern of expression of NRG3 was observed in RNA blots frommurine tissues using a probe derived from the region of murine NRG3 thatoverlaps the EGF-like domain.

[0288] The tissue distribution of NRG3 expression was characterized byin situ hybridization using tissues of embryonic and adult mice. Atembryonic day 13 (E13) (the earliest time point examined), NRG3 mRNA wasconfined to the nervous system. A strong signal for NRG3 mRNA in thebrain, spinal cord, trigeminal, vestibular-cochlear and spinal gangliaof embryonic day 16 (E16) mice was also demonstrated. Regions of thetelencephalon containing differentiating cells (e.g., the corticalplate) displayed an intense NRG3 signal, whereas the underlying regionscontaining proliferating or migrating cells (ventricular andsubventricular zones), showed little expression. Thus, NRG3 appeared tobe expressed mainly in the nervous system of embryonic mice. In adultanimals NRG3 antisense probes hybridized to mRNA in spinal cord andnumerous brain regions including deep cerebellar nuclei, vestibularnuclei, cerebral cortex, piriform cortex, anterior olfactory nucleus,medial habenula, hippocampus, hypothalamus and thalamus.

Example 4 Characterization of the Binding Characteristics of NRG3Fragments

[0289] A. Expression and Purification of NRG3^(EGF) Fusion Protein inMammalian Cells

[0290] To examine the binding characteristics of the NRG3^(EGF)-likedomain as well as to demonstrate the functionality of an NRG3 fragmentof the invention, a soluble fusion protein was prepared comprising asequence of EGF-like domain, which domain has the same amino acidsequence in mouse and human NRG3.

[0291] A secreted, epitope tagged polypeptide comprising the EGF-likedomain of murine NRG3₂₈₄₋₃₄₄ was constructed by linking in the expressedN-terminal to C-terminal direction 1) the coding sequence for the gDsignal sequence and epitope tag (Mark, M. R. et al. (1994) J Biol. Chem.269, 10720-10728); 2) the sequences encoding amino acids 284-344 ofmurine NRG3 (identical to human NRG3 amino acids 282 to 342); and 3) thecoding sequences of the Fc portion of human IgG₁ in pSAR.SD5 vector(psar.SD5, from A. Shen, Genentech, Inc.). The plasmid encoding thesesequences was designated NRG3^(EGF).Fc. The NRG3^(EGF).Fc expressionplasmid was transfected using LipofectAMINE (GIBCO/BRL, Bethesda, Md.)into DHFR Chinese hamster ovary cells (CHO/DP12; ATCC designation CCL9096). Clones were selected in glycine/hypoxanthine/thymidine minusmedium see, for example, (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory (1989)), pooled, andexpanded. The encoded fusion protein was expressed in cultures of theselected clones. Conditioned media from these cells were collected andthe recombinant protein purified by a HiTrap protein A affinity column(Pharmacia).

[0292] A monomeric fusion protein designated NRG3^(EGF).H6 fusionprotein was produced in the same system as the Fc-fusion protein andpurified through a cobalt affinity column. NRG3^(EGF).H6 comprises theN-terminal gD tag, murine NRG3₂₈₄₋₃₄₄, and a coding sequence for sixhistidine residues. Purification was based on the affinity of thehistidine side chains for immobilized cobalt using a cobalt affinitycolumn (Cobalt affinity column, R. Vandlen, Genentech, Inc.). Proteinconcentration was determined by BioRad Protein Assay (BioRad, Richmond,Calif.).

[0293] B. Generation of K562^(erbB) Cell Lines.

[0294] Stable cell lines that expressed human ErbB2, ErbB3 or ErbB4receptors were derived from K562 cells (K562 cells have ATCC designationCCL 243). cDNAs of human erbB2, erbB3 and erbB4 were from L. Bald and G.Scoffer, Genentech (Sliwkowski, M. et al. (1994), J. Biol Chem.269:14661-14665). These cDNAs were subcloned into CMV-based expressionvectors and introduced into the K562 human myeloid leukemia cell line byelectroporation (1180 mF, 350 V). The transfectants were cultured inRPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100U/ml penicillin, 100 mg/ml streptomycin, and 10 mM HEPES containing 0.8mg/ml G418. Resistant clones were obtained by limiting dilution, andreceptor expression was confirmed by western blot and NRG bindingassays. Receptor expression was confirmed by western blot analysis usingantibodies for each of the ErbB receptors (antibodies prepared atGenentech, Inc.) Phorbol ester stimulation was found to significantlyenhance receptor expression in both the ErbB3 and ErbB4 transfectants,and the stably transfected K562 cell lines were cultured in mediumcontaining 10 ng/ml Phorbol, 12-Myristate, 12-Acetate- (PMA) overnightprior to use.

[0295] C. FACS Analysis.

[0296] For each binding reaction, 5×10⁵ stably transfected K562 cellswere suspended in PBS/2%BSA at 4° C. for 30 min followed by incubationwith 5 μg of isolated, purified NRG3^(EGF).Fc (MW 90 kDa) in a volume of0.25 ml on ice for 60 min. 1 μg of primary antibody (anti-gD oranti-ErbB receptor) and secondary PE-conjugated (CALTAG, CA., goat 15-anti-mouse, 1:100 dilution) antibodies were added sequentially with30-60 min incubation time and extensive washes before each addition.FACS analyses were performed on a Becton & Dickson FACS instrument.Anti-gD (5B6), anti-ErbB2 receptor (4D5), anti-ErbB3 receptor (2F9) andanti-ErbB4 receptor (3B9) monoclonal antibodies were prepared usingstandard techniques by the Monoclonal Antibody Group, Genentech, Inc.

[0297] D. The EGF-Like Domain of NRG3 Binds Specifically to the ErbB4Receptor Tyrosine Kinase.

[0298] To identify the receptor(s) for NRG3, the ability of NRG3 to bindto any of the known neuregulin receptors was investigated. Stable celllines were generated which expressed receptors ErbB2, ErbB3, or ErbB4.The parental cell line K562 does not express detectable levels of ErbBreceptors (FIG. 6A). K562^(erb2), K562^(erb3) and K562^(erb4) cellsexpressed only the corresponding receptors (FIGS. 6B-6D).

[0299] Since the EGF-like domain determines the binding specificity ofNRG1 to their receptors, a protein containing an epitope tagged versionof the EGF-like domain of NRG3 fused to the Fc portion of human IgG wasexpressed and purified. Using a FACS assay, it was observed thatNRG3^(EGF).Fc bound to cells expressing ErbB4 receptor (FIG. 6H).Binding was specific in that NRG3^(EGF).Fc did not bind to either theparental K562 cells, or cells expressing either ErbB2 or ErbB3 (FIG.6E-6G). A control fusion protein, RSE.Fc, did not bind to any of thesecell lines. This binding of NRG3^(EGF).Fc to K562^(erb4) cells wascompeted in a dose-dependent fashion by the EGF-like domain ofheregulin-β1 (NRG1^(EGF)), but not by RSE.Fc, suggesting thatNRG3^(EGF).Fc interacts directly with ErbB4 receptors on the cellsurface.

[0300] A soluble form of the ErbB4 receptor was co-immunoprecipitated byNRG3^(EGF).Fc in vitro, further demonstrating the binding ofNRG3^(EGF).Fc to ErbB4 receptor.

[0301] The binding of NRG3^(EGF).Fc to ErbB4 receptor was furtheranalyzed by direct competitive binding assays using ¹²⁵I-labeledNRG3^(EGF).Fc. Purified NRG3^(EGF).Fc was radio-iodinated using thelactoperoxidase method as described by Sliwkowski et al. (Sliwkowski, M.X. et al. (1994) J Biol. Chem. 269, 14661-5). The average specificactivity of the radiolabeled protein was 300 μCi/μg. Binding of¹²⁵I-NRG3^(EGF).Fc to immobilized ErbB4.Fc was competed by eitherNRG3^(EGF).Fc or EGF domain of NRG1^(EGF) (rHRGβ1₁₇₇₋₂₄₄) in aconcentration dependent manner.

[0302] The displacement binding assays were performed in Maxisorp C96-wells (Nunc, Naperville, Ill.). Goat anti-human antibody (BoehringerMannheim, Germany) was coated on the plate at a concentration of 0.2μg/well in 100 μl of 50 mM sodium carbonate buffer (pH 9.6) at 4° C.,overnight. The plate was blocked by 1%BSA in TBST buffer (25 mM Tris, pH7.5, 150 mM NaCl, 0.02% Tween 20) for 30 min at room temperature (RT). Asoluble form of ErbB4 receptor was added at 15 ng/well in 1%BSA/TBST andincubated for 1.5 hr at RT. To prevent radiolabeled protein frominteracting with residual goat anti-human antibodies, 1 μM of ahumanized monoclonal antibody (rhuMAB HER2; Carter, P. et al. (1992)Proc. Natl. Acad. Sci. USA 89:4285-9) was added to the plate for 20 minand was included in the subsequent binding reaction.

[0303] The competitive binding assay was then initiated by the additionof 80 pM (200,000 cpm) of ¹²⁵I-NRG3^(EGF).Fc along with variousconcentrations of unlabeled NRG3^(EGF).Fc or NRG1^(EGF) (E.coli-expressed, without Fe). NRG1^(EGF) is the EGF domain of NRG1,corresponding to amino acids 177-244 of the neuregulin-β1 isoform(Holmes, W. E. et al. (1992) Science 256:1205-10) and obtained from J.A. Lofgren, Genentech, Inc. The final incubation volume was 100 μl inbinding buffer (F-12/DMEM medium, 50 mM HEPES, pH7.5, 2%BSA) and thereaction was allowed to proceed at RT for 1.5 hr. The unbound materialwas washed by TBST extensively, and the bound radioactivity was countedon a Beckman IsoData gamma-counter (Smith-Kline Beckman, Pa.). Data wasanalyzed using a nonlinear regression computer program.

[0304] Based on the results of the binding experiments as shown in FIGS.6A-6H, the estimated affinity (K_(i)) for NRG3^(EGF).Fc for binding toErbB4.Fc was determined to be 9±4 nM (n=4), and the apparent k_(I) ofNRG1^(EGF) was approximately 1 nM. The shallowness of the displacementcurve of NRG3^(EGF).Fc may be due to the fact that the NRG3^(EGF).Fc isexpressed as a bivalent Fc fusion protein (FIG. 7). The results of thecontrol experiments showed that ¹²⁵I-NRG3^(EGF).Fc did not bind controlreceptor RSE.Fc in the same experiment, and RSE.Fc did not compete¹²⁵I-NRG 3^(EGF).Fc bound to ErbB4.Fc.

[0305] E. Tyrosine Phosphorylation Assay.

[0306] NRG1 binds and activates ErbB2, ErbB3 and ErbB4 receptorresulting in tyrosine phosphorylation and downstream signaling events(Sliwkowski, M. X., et al. (1994), supra; Plowman, G. D. et al. (1993)supra; and Carraway, K. L. and Cantley, L. C. (1994), surpa). Asdemonstrated in the preceding example, NRG3 binds ErbB4 receptor, butnot ErbB2 or ErbB3 receptors at a detectable level. The ability of theEGF-like domain of NRG3 (NRG3EGF) to activate ErbB4 receptor,K562^(erbB4) cells was examined.

[0307] K562^(erbB4) cells or MDA-MB-453 cells (negative control; ATCCdesignation HB 131) were cultured in medium lacking serum for 12 hoursand then stimulated with NRG3^(EGF).Fc, NRG^(EGF).H6 or NRG1EGF.K562^(erbB4) cells were treated with 2.5 nM or 25 nM of NRG3^(EGF).Fcfor 3 min or 8 min. As a positive control, the cells were similarlytreated with NRG1^(EGF).

[0308] ErbB4 receptor tyrosine phosphorylation was detected byimmunoprecipitation and Western blot according to the followingprocedure. Cells were lysed with lysis buffer (20 mM Tris, pH 7.5, 100mM NaCl, 30 mM NaF, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 1% Triton X-100, 2mM sodium vanadate, 2 mM sodium molybdate, 2 mM of PMSF). After removingcell debris by centrifugaton, 1 μg of anti-ErbB4 receptor monoclonalantibody (C-18, Santa Cruz Biotechnology, Santa Cruz, Calif.) was addedtogether with 20 μl of protein A-agarose slurry (Sigma, St. Louis, Mo.).Immunoprecipitation was performed at 4° C. overnight, complexes werecollected by centrifugation and washed three times with 1 ml lysisbuffer. Proteins were separated by reducing SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) on Novex 4%-12% minigels and transferred tonitrocellulose. The blots were probed with peroxidase conjugatedanti-phosphotyrosine antibody (Transduction Laboratory). The blot wasstripped and reprobed with anti-ErbB4 receptor antibody followed byperoxidase conjugated goat anti-rabbit IgG antibody (Sigma) to visualizeErbB4 receptor proteins.

[0309] Based on these experiments, it was demonstrated thatNRG3^(EGF).Fc stimulated ErbB4 receptor tyrosine phosphorylation at bothtime points and in a dose dependent manner.

[0310] To confirm the ability of NRG3^(EGF) to activate the ErbB4receptor tyrosine phosphorylation, receptor activation in the humanbreast cancer cell line MDA-MB-453 was examined. This cell lineexpresses high level of ErbB2 and ErbB3 receptors, and low levels ofErbB4 receptor. Treatment of MDA-MB-453 cells with NRG3^(EGF).Fc or witha monomeric form of the EGF domain (NRG3^(EGF).H6) resulted insubstantial increase of tyrosine phosphorylation of ErbB4 receptor.

[0311] NRG family members and other members in the EGF family display acomplex pattern of receptor binding. In most cases, one ligand is ableto bind several combinations of receptor homo- and heterodimers(Karunagaran, D. et al. (1996) EMBO J 15:254-264,Beerli, R. R. andHynes, N. E. (1996) J. Biol. Chem. 271:6071-6076). For example, NRGsbind ErbB2/ErbB3 receptor heterodimers and ErbB4/ErbB4 receptorhomodimers with high affinity but ErbB3/ErbB3 receptor homodimers withlow affinity (Sliwkowski, M. X. et al. (1994) J. BioL Chem. 269,14661-5, Carraway, K. L. and Cantley, L. C. (1994) Cell 78, 5-8, Tzahar,E. et al. (1994) J BioL Chem. 269, 25226-33, Carraway, K. L. r. et al.(1994) J Biol. Chem. 269, 14303-6, and Kita, Y. A. et al. (1994) FEBSLett. 349, 139-43). Betacellulin binds both EGFR and ErbB4 homodimers(Riese, D. J. et al. (1995) Mol. Cell. Biol. 15:5770-6). The EGF-likedomains of EGF and NRG1 family members determine the specificity ofreceptor activation (Barbacci, E. G. et al. (1995) J. Biol. Chem.270:9585-9589). The limited amino acid sequence homology in the EGF-likedomains of NRG3 and NRG1 suggests that NRG3 may have a differentspectrum of receptor interactions relative to members of the NRG family,but with potentially overlapping binding sites, since binding of theEGF-like domain of NRG3 to ErbB4 can be competed by the EGF-like domainof NRG1.

[0312] NRG3^(EGF).Fc did not bind to K562 cells that express eitherErbB2 or ErbB3 (FIG. 6E-6G), or to MDA-MB-486 cells which express highlevels of the EGFR. An increase in phosphorylation of either the EGFR,ErbB2 or ErbB3 in MDA-MB-453 cells treated with NRG3 also was notobserved.

[0313] Most variants of NRGs, with the exception of the neural specificform of SMDF, are widely expressed in numerous tissues including brain,heart, skeletal muscle, breast, liver, lung, among others. Betacellulin,a ligand for both EGFR and ErbB4, also displays broad tissue expressionpatterns (Shing, Y. et al. (1993) Science 259, 1604-7; Sasada, R. et al.(1993) Biochem. Biophy. Res. Com. 190, 1173-9). In contrast, theexpression of NRG3 is strikingly restricted to neural tissues asdisclosed herein by Northern analysis and in situ hybridization.Developmentally, NRG3 mRNA can be detected as early as E11 (but not E4)in mouse as judged by Northern blot and E13 by in situ hybridization(the earliest age examined). ErbB4 is predominantly expressed in brain,heart and skeletal muscle (Plowman, G. D. et al. (1993) Proc. Natl.Acad. Sci. USA 90, 1746-50). ErbB4 was also shown to be broadlydistributed in the brains of chick embryos (E14, E17, predominantly inneurons) (Francoeur, J. R. et al. (1995) J. Neur. Res. 41, 836-45), inrat retina cultures (Bermingham-McDonogh, 0. et al. (1996) Development122, 1427-38.), at neuromuscular synapses (Zhu, X. et al. (1995) EMBO J.14, 5842-8.), but not in cultured human and rat Schwann cells (Grinspan,J. B. et al. (1996) J. Neuroscience 16, 6107-6118,Levi, A. D. et al.(1995) J Neuroscience 15, 1329-40.). Recently, ErbB4 was found toco-localize with GABA⁺cells (Weber, J. et al. (1996) Soc Neurosci Abstr22, 1579.). Thus, the same receptor may mediate distinct biologicalfunctions in different tissues or cell types when interacted withcorresponding tissue-specific ligands. For example, NRG1may serve as aligand for ErbB4 during heart development, betacellulin may act as amitogenic ligand for ErbB4 in variety of cell types, while neuralspecific ligand(s) (such as NRG3) may function as trophic or guidancemolecules on ErbB4 receptor expressing cells in the central orperipheral nervous systems.

Example 5 Binding and ErbB4 Receptor Tyrosine Kinase Activation by FullLength Mouse and Human NRG3s.

[0314] A full length murine NRG3 or human NRG3 was synthesized based onthe murine and human consensus nucleic acid sequences SEQ ID NO: 1 andSEQ ID NO: 5, respectively and the NRG3s were expressed as amino acidsequences. Based on the experiments described herein for thecharacterization of NRG3-EGF binding and ErbB4 receptor activation,analogous experiments are performed for the full length consensus NRG3from mouse and human sources. Adjustments to the reaction conditions aremade to optimize pH, solutes and their concentrations, and otherrelevant parameters to allow ErbB4 receptor-binding of the full lengthconsensus NRG3 and ErbB4 receptor activation.

[0315] Alternatively, a murine or human NRG3 polypeptide fragmentcomprising the EGF-like domain but lacking the transmembrane domain issynthesized and tested for ErbB4 receptor binding and activation asdescribed herein. Such a NRG3 fragment may, for example, include theextracellular domain of a NRG3, which extracellular domain contains theEGF-like domain.

[0316] A NRG3 extracellular domain may optionally be fused to animmunoglobulin constant region, as shown herein for the NRG3-EGF-Fcfusion proteins. As an Fc fusion protein, the NRG3 extracellulardomain-Fc protein is expected to form a dimer. The immunoglobulinconstant region is preferably from IgG, but may also be taken from IgM,IgA and IgE and remain within the scope of the invention.

[0317] Where a monomeric fusion protein is desired that retains bindingactivity or binding and activation ability, the extracellular domain isfused to, for example, a series of histidine residues as disclosedherein for the NRG3-EGF-H6 immunoadhesion.

[0318] Adjustments to the binding reaction conditions are made tooptimize pH, solutes and their concentrations and other relevantparameters to allow ErbB4 receptor-binding of the NRG3 fragment andErbB4 receptor activation.

Example 6 Enhancement of Cellular Proliferation

[0319] Enhancement of cellular proliferation is exemplified by thefollowing assay in which cells expressing ErbB4 receptor on theirsurface are treated with NRG3. It is understood that according to theinvention, the cells may be treated with a NRG3 fragment (such as theNRG3 EGF-like domain) or a NRG3 variant.

[0320] As an example, rat retina cells which naturally express ErbB4receptor (Bermingham-McDonogh, O. et al. (1996) Development 122,1427-38) are cultured by standard techniques. The cultured cells arecontacted with NRG3 in a dose dependent manner and an increase in cellnumber (e.g. a 30% percent increase at 48 hours) and EC50 is determined.

[0321] NRG3 treatment may also alter the morphology of these cells;untreated cells were multipolar with numerous branched processes whereasNRG3-treated cells may become spindle-shaped smooth processes and/oralign themselves in a parallel array.

[0322] NRG3 is believed to stimulate neuronal cell growth in a dosedependent manner. NRG3 alone is expected to produce a significantincrease in neuronal cell number compared to control medium. Asynergistic effect may be observed between other neuronal proliferationenhancers such as gas6 (growth arrest-specific gene; see, for example,Schneider et al., Cell 54:787-793 (1988); and Manfioletti et al. inMolec. Cell Biol. 13(8):4976-4985 (1993)) and/or heregulin. NRG3 isexpected to increase both cell number and thymidine incorporation asmeasures of cell proliferation.

[0323] NRG3 is expected to have an effect on cell morphology asdetermined by viewing phase contrast micrographs of ErbB4receptor-expressing neuronal cells grown in various media containingNRG3 alone or NRG3 plus other cell proliferation enhancing compoundssuch as heregulin, gas6, fetal bovine serum, and the like.Photomicrographs are taken after 96 hours of culture. The cells grown inthe presence of NRG3 are expected to have processes which are notobserved in cells grown in the absence of NRG3.

[0324] Cells are stained by immunofluorescence for markers specific forthe cultured neuronal cells. Briefly, passaged ErbB4 receptor-expressingneuronal cells are contacted with NRG3 and cultured for 24 hours onlaminin coated Chamber slides and fixed in 10% formalin in PBS. Fixedcells are blocked with 10% goat serum and incubated with rabbit derivedanti-marker antibody at dilutions recommended by the distributor.Specific binding of the primary antibody is observed by staining withgoat anti-rabbit IgG (Fab′)₂-FITC conjugates. Cells are counter-stainedwith DNA dye propidium iodide. Negative controls are run on WI-38 cellswhich stain negative. Cells grown under these conditions are expected toshow 100% immunofluorescent staining for the cell markers.

[0325] The ability of NRG3 to stimulate proliferation in ErbB4receptor-expressing neuronal cells through the ErbB4 tyrosine kinasereceptors may be investigated as follows. Cells are stimulated withvarious amounts of NRG3 (for example, 0 to 200 nM) for 15 min in a 37°C. incubator. Cell lysates are prepared and immunoprecipitated with ananti-ErbB4 receptor antibody. Tyrosine phosphorylation of ErbB4 receptoris detected with 4G10 anti-phosphorylation antibody. Approximately 10⁶cells are grown to near confluence in defined media. Cells are treatedwith NRG3 for 15 min in a 37° C. incubator and lysed on ice with 1 ml oflysis buffer (20 mM HEPES, pH7.4, 135 mM NaCl, 50 mM NaF, 1 mM sodiumvanadate and 1 mM sodium molybdate, 2 mM EDTA and 2 mM EGTA, 10%glycerol, 1%NP40, 1 μM okadaic acid, 1 mM PMSF and 1 mM AEBSF). Celllysates are clarified by centrifuging at 14000× g at 4° C. for 10 min.Immunoprecipitations are performed using approximately 1 μg of rabbitanti-ErbB4 receptor antibody or 2 μl of rabbit anti-ErbB4 receptorantiserum. Immunocomplexes are collected with 10 μl of Protein ASepharose CL-4B beads. Proteins are separated on Novex 4-12% gradientgel and transferred onto nitrocellulose membrane. Anti-phosphotyrosineimmunoblots are performed using 4G10 mouse anti-phosphotyrosine antibody(UBI), goat anti-mouse horseradish peroxidase conjugate and ECLdeveloping kit (Amersham). Addition of NRG3 to ErbB4 receptor-expressingneuronal cells is expected to cause autophosphoralation of ErbB4receptor tyrosine residue(s).

[0326] It is beneficial to have populations of mammalian neuronal cells(preferably human cells) for use as cellular prostheses fortransplantation into areas of damaged spinal cord in an effort toinfluence regeneration of interrupted central axons, for assisting inthe repair of peripheral nerve injuries and as alternatives to multipleautografts. See Levi et al., J. Neuroscience 14(3):1309-1319 (1994). Theuse of cell culture techniques to obtain an abundant source ofautologous graft material from a small biopsy has already met withclinical success in providing human epidermal cells to cover extensiveburns (Gallico et al, N. Eng J. Med. 311:338-451(1984)). Accordingly, itis expected that the above approach will meet with success in mammals,including humans.

[0327] All documents cited throughout the specification as well as thereferences cited therein are hereby expressly incorporated by referencein their entirety. While the present invention is illustrated withreference to specific embodiments, the invention is not so limited. Itwill be understood that further modifications and variations arepossible without diverting from the overall concept of the invention.All such modifications are intended to be within the scope of thepresent invention.

[0328] Deposit of Material

[0329] The following materials have been deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC): ATCC Dep. Material No. Deposit Date mouse NRG3 pLXSN.mNRG3209156 July 22, 1997 human NRG3B1 pRK5.tk.neo.hNRG3B1 209157 July 22,1997 human NRG3B2 pRK5.tk.neo.hNRG3B2 209297 September 23, 1997

[0330] These deposits are made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposit will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC § 122 and the Commissioner's rulespursuant thereto (including 37 CFR §1.14 with particular reference to886 OG 638).

[0331] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

[0332] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1 23 2538 base pairs Nucleic Acid Single Linear mouse NRG3 nucleic acid1-2538 1 CCTGACCGGC CGGCGGCGCC CGGGCCGGTC TCGCCCCTCT ACCGAGCGCC 50TCGCCGCCCC CTCCCCGGCC CGCGTCCCCT CCCCCGTCCT CTCCTCCCCG 100 CCCGCCGCCCGCCTCTCGGG GGGAGGGGCG TGGGGGCAGG GAGCCGATTT 150 GCATGCGGCC GCCGCGGCCGCTGCCTGAGC CGGAGCCCGC CGCCGCCGGA 200 GCCCGCGCCC GCGCCCGCGC CCGGCCCGCGCGGCCCCATG CCTCTGGCGC 250 GGCCCTCGGG GGGGCGAAGG TGAAGATCGG CTCCTAGGATGAGTGAAGGG 300 GCGGCCGGTG CCTCGCCACC TGGTGCCGCT TCGGCAGCCG CCGCCTCAGC350 CGAGGAGGGC ACCGCGGCGG CTGCGGCGGC GGCGGCGGCG GGCGGGGGCC 400CGGACGGCGG CGGAGAAGGG GCGGCCGAAC CCCCCCGGGA GTTACGCTGT 450 AGCGACTGCATCGTGTGGAA CCGGCAGCAG ACGTGGTTGT GCGTGGTGCC 500 TCTGTTCATC GGCTTCATCGGCCTGGGGCT CAGCCTCATG CTGCTTAAAT 550 GGATCGTGGT AGGCTCCGTC AAGGAGTACGTGCCCACGGA CCTGGTGGAC 600 TCCAAGGGAA TGGGCCAGGA CCCCTTCTTC CTCTCCAAGCCCAGCTCTTT 650 CCCCAAGGCT ATGGAAACCA CCACAACAAC CACTTCTACC ACGTCCCCCG700 CCACCCCCTC TGCCGGCGGC GCCGCTTCTT CCAGGACGCC TAACCGGATT 750AGCACCCGCT TGACCACCAT CACACGGGCA CCCACCCGCT TCCCTGGGCA 800 CCGGGTTCCCATCCGGGCTA GCCCGCGCTC TACCACAGCA CGGAACACTG 850 CTGCCCCTCC GACGGTCCTGTCCACCACGG CCCCTTTCTT CAGTAGCAGC 900 ACGCCCGGCT CCCGACCCCC GATGCCAGGAGCCCCCAGTA CGCAGGCGAT 950 GCCTTCCTGG CCCACTGCGG CGTATGCTAC CTCCTCCTACCTCCACGATT 1000 CCACTCCCTC CTGGACCCTG TCACCCTTTC AGGATGCTGC TGCCGCCTCT1050 TCCTCCTCAC CCTCTTCCAC CTCCTCCACT ACCACCACCC CAGAAACTAG 1100CACCAGCCCC AAATTTCATA CTACAACATA CTCCACTGAA CGATCTGAGC 1150 ACTTCAAACCCTGTCGAGAC AAGGACCTGG CGTATTGTCT CAATGATGGT 1200 GAATGCTTTG TGATTGAGACCCTGACAGGA TCCCATAAGC ACTGTCGGTG 1250 CAAGGAAGGC TACCAAGGAG TCCGTTGTGATCAATTTCTG CCGAAAACAG 1300 ACTCCATCTT ATCGGATCCA ACAGACCACT TGGGGATTGAATTCATGGAG 1350 AGTGAAGACG TTTATCAAAG GCAGGTGCTG TCAATTTCAT GTATCATCTT1400 TGGAATTGTC ATCGTGGGCA TGTTCTGTGC AGCATTCTAC TTCAAAAGCA 1450AGAAACAAGC TAAACAAATT CAGGAGCACC TGAAAGAGTC ACAGAATGGG 1500 AAGAACTACAGCCTCAAGGC ATCCAGCACA AAGTCTGAGA GCTTGATGAA 1550 GAGCCATGTC CATCTACAAAATTATTCAAA GGCGGATAGG CATCCTGTGA 1600 CTGCGCTGGA GAAAATAATG GAGTCAAGTTTTTCAGCTCC CCAGTCGTTC 1650 CCAGAAGTCA CTTCTCCTGA CCGAGGAAGC CAGCCTATCAAGCACCACAG 1700 CCCAGGACAA AGGAGTGGGA TGTTGCATAG GAATACTTTC AGAAGGGCAC1750 CACCCTCACC CCGAAGTCGA CTGGGTGGTA TTGTAGGACC AGCATATCAA 1800CAACTTGAAG AATCAAGAAT TCCAGACCAG GATACGATAC CTTGCCAAGG 1850 GATAGAGGTCAGGAAGACTA TATCCCACCT GCCTATACAG CTGTGGTGTG 1900 TTGAAAGACC CCTGGACTTAAAGTATGTGT CCAATGGCTT AAGAACCCAA 1950 CAAAATGCAT CAATAAATAT GCAACTGCCTTCAAGAGAGA CAAACCCCTA 2000 TTTTAATAGC TTGGATCAAA AGGACCTGGT GGGTTATTTATCCCCAAGGG 2050 CCAATTCTGT GCCCATCATC CCGTCGATGG GTCTAGAAGA AACCTGCATG2100 CAAATGCCAG GGATTTCTGA CGTCAAAAGC ATTAAATGGT GCAAAAACTC 2150CTACTCCGCT GACATTGTCA ACGCGAGTAT GCCAGTCAGT GATTGTCTTC 2200 TAGAAGAACAACAGGAAGTG AAAATATTAC TAGAGACTGT GCAGGAACAG 2250 ATCCGGATTC TGACTGATGCCAGACGGTCA GAAGACTTCG AACTGGCCAG 2300 CATGGAAACT GAGGACAGTG CGAGCGAAAACACAGCCTTT CTCCCCCTGA 2350 GTCCCACGGC CAAATCAGAA CGAGAGGCAC AATTTGTCTTAAGAAATGAA 2400 ATACAAAGAG ACTCTGTGCT AACCAAGTGA CTGGAAATGT AGGAATCTGT2450 GCATTATATG CTTTGCTAAA CAGGAAGGAG AGGAAATTAA ATACAAATTA 2500TTTATATGCA TTAATTTAAG AGCATCTACT TAGAAGCC 2538 713 amino acids AminoAcid Linear Mouse NRG3 (mNRG3)/amino acid seq. 1-713 2 Met Ser Glu GlyAla Ala Gly Ala Ser Pro Pro Gly Ala Ala Ser 1 5 10 15 Ala Ala Ala AlaSer Ala Glu Glu Gly Thr Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala GlyGly Gly Pro Asp Gly Gly Gly Glu Gly Ala 35 40 45 Ala Glu Pro Pro Arg GluLeu Arg Cys Ser Asp Cys Ile Val Trp 50 55 60 Asn Arg Gln Gln Thr Trp LeuCys Val Val Pro Leu Phe Ile Gly 65 70 75 Phe Ile Gly Leu Gly Leu Ser LeuMet Leu Leu Lys Trp Ile Val 80 85 90 Val Gly Ser Val Lys Glu Tyr Val ProThr Asp Leu Val Asp Ser 95 100 105 Lys Gly Met Gly Gln Asp Pro Phe PheLeu Ser Lys Pro Ser Ser 110 115 120 Phe Pro Lys Ala Met Glu Thr Thr ThrThr Thr Thr Ser Thr Thr 125 130 135 Ser Pro Ala Thr Pro Ser Ala Gly GlyAla Ala Ser Ser Arg Thr 140 145 150 Pro Asn Arg Ile Ser Thr Arg Leu ThrThr Ile Thr Arg Ala Pro 155 160 165 Thr Arg Phe Pro Gly His Arg Val ProIle Arg Ala Ser Pro Arg 170 175 180 Ser Thr Thr Ala Arg Asn Thr Ala AlaPro Pro Thr Val Leu Ser 185 190 195 Thr Thr Ala Pro Phe Phe Ser Ser SerThr Pro Gly Ser Arg Pro 200 205 210 Pro Met Pro Gly Ala Pro Ser Thr GlnAla Met Pro Ser Trp Pro 215 220 225 Thr Ala Ala Tyr Ala Thr Ser Ser TyrLeu His Asp Ser Thr Pro 230 235 240 Ser Trp Thr Leu Ser Pro Phe Gln AspAla Ala Ala Ala Ser Ser 245 250 255 Ser Ser Pro Ser Ser Thr Ser Ser ThrThr Thr Thr Pro Glu Thr 260 265 270 Ser Thr Ser Pro Lys Phe His Thr ThrThr Tyr Ser Thr Glu Arg 275 280 285 Ser Glu His Phe Lys Pro Cys Arg AspLys Asp Leu Ala Tyr Cys 290 295 300 Leu Asn Asp Gly Glu Cys Phe Val IleGlu Thr Leu Thr Gly Ser 305 310 315 His Lys His Cys Arg Cys Lys Glu GlyTyr Gln Gly Val Arg Cys 320 325 330 Asp Gln Phe Leu Pro Lys Thr Asp SerIle Leu Ser Asp Pro Thr 335 340 345 Asp His Leu Gly Ile Glu Phe Met GluSer Glu Asp Val Tyr Gln 350 355 360 Arg Gln Val Leu Ser Ile Ser Cys IleIle Phe Gly Ile Val Ile 365 370 375 Val Gly Met Phe Cys Ala Ala Phe TyrPhe Lys Ser Lys Lys Gln 380 385 390 Ala Lys Gln Ile Gln Glu His Leu LysGlu Ser Gln Asn Gly Lys 395 400 405 Asn Tyr Ser Leu Lys Ala Ser Ser ThrLys Ser Glu Ser Leu Met 410 415 420 Lys Ser His Val His Leu Gln Asn TyrSer Lys Ala Asp Arg His 425 430 435 Pro Val Thr Ala Leu Glu Lys Ile MetGlu Ser Ser Phe Ser Ala 440 445 450 Pro Gln Ser Phe Pro Glu Val Thr SerPro Asp Arg Gly Ser Gln 455 460 465 Pro Ile Lys His His Ser Pro Gly GlnArg Ser Gly Met Leu His 470 475 480 Arg Asn Thr Phe Arg Arg Ala Pro ProSer Pro Arg Ser Arg Leu 485 490 495 Gly Gly Ile Val Gly Pro Ala Tyr GlnGln Leu Glu Glu Ser Arg 500 505 510 Ile Pro Asp Gln Asp Thr Ile Pro CysGln Gly Ile Glu Val Arg 515 520 525 Lys Thr Ile Ser His Leu Pro Ile GlnLeu Trp Cys Val Glu Arg 530 535 540 Pro Leu Asp Leu Lys Tyr Val Ser AsnGly Leu Arg Thr Gln Gln 545 550 555 Asn Ala Ser Ile Asn Met Gln Leu ProSer Arg Glu Thr Asn Pro 560 565 570 Tyr Phe Asn Ser Leu Asp Gln Lys AspLeu Val Gly Tyr Leu Ser 575 580 585 Pro Arg Ala Asn Ser Val Pro Ile IlePro Ser Met Gly Leu Glu 590 595 600 Glu Thr Cys Met Gln Met Pro Gly IleSer Asp Val Lys Ser Ile 605 610 615 Lys Trp Cys Lys Asn Ser Tyr Ser AlaAsp Ile Val Asn Ala Ser 620 625 630 Met Pro Val Ser Asp Cys Leu Leu GluGlu Gln Gln Glu Val Lys 635 640 645 Ile Leu Leu Glu Thr Val Gln Glu GlnIle Arg Ile Leu Thr Asp 650 655 660 Ala Arg Arg Ser Glu Asp Phe Glu LeuAla Ser Met Glu Thr Glu 665 670 675 Asp Ser Ala Ser Glu Asn Thr Ala PheLeu Pro Leu Ser Pro Thr 680 685 690 Ala Lys Ser Glu Arg Glu Ala Gln PheVal Leu Arg Asn Glu Ile 695 700 705 Gln Arg Asp Ser Val Leu Thr Lys 710713 362 amino acids Amino Acid Linear mNRG3 extracellular domainAminoacid seq 1-362 3 Met Ser Glu Gly Ala Ala Gly Ala Ser Pro Pro Gly Ala AlaSer 1 5 10 15 Ala Ala Ala Ala Ser Ala Glu Glu Gly Thr Ala Ala Ala AlaAla 20 25 30 Ala Ala Ala Ala Gly Gly Gly Pro Asp Gly Gly Gly Glu Gly Ala35 40 45 Ala Glu Pro Pro Arg Glu Leu Arg Cys Ser Asp Cys Ile Val Trp 5055 60 Asn Arg Gln Gln Thr Trp Leu Cys Val Val Pro Leu Phe Ile Gly 65 7075 Phe Ile Gly Leu Gly Leu Ser Leu Met Leu Leu Lys Trp Ile Val 80 85 90Val Gly Ser Val Lys Glu Tyr Val Pro Thr Asp Leu Val Asp Ser 95 100 105Lys Gly Met Gly Gln Asp Pro Phe Phe Leu Ser Lys Pro Ser Ser 110 115 120Phe Pro Lys Ala Met Glu Thr Thr Thr Thr Thr Thr Ser Thr Thr 125 130 135Ser Pro Ala Thr Pro Ser Ala Gly Gly Ala Ala Ser Ser Arg Thr 140 145 150Pro Asn Arg Ile Ser Thr Arg Leu Thr Thr Ile Thr Arg Ala Pro 155 160 165Thr Arg Phe Pro Gly His Arg Val Pro Ile Arg Ala Ser Pro Arg 170 175 180Ser Thr Thr Ala Arg Asn Thr Ala Ala Pro Pro Thr Val Leu Ser 185 190 195Thr Thr Ala Pro Phe Phe Ser Ser Ser Thr Pro Gly Ser Arg Pro 200 205 210Pro Met Pro Gly Ala Pro Ser Thr Gln Ala Met Pro Ser Trp Pro 215 220 225Thr Ala Ala Tyr Ala Thr Ser Ser Tyr Leu His Asp Ser Thr Pro 230 235 240Ser Trp Thr Leu Ser Pro Phe Gln Asp Ala Ala Ala Ala Ser Ser 245 250 255Ser Ser Pro Ser Ser Thr Ser Ser Thr Thr Thr Thr Pro Glu Thr 260 265 270Ser Thr Ser Pro Lys Phe His Thr Thr Thr Tyr Ser Thr Glu Arg 275 280 285Ser Glu His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala Tyr Cys 290 295 300Leu Asn Asp Gly Glu Cys Phe Val Ile Glu Thr Leu Thr Gly Ser 305 310 315His Lys His Cys Arg Cys Lys Glu Gly Tyr Gln Gly Val Arg Cys 320 325 330Asp Gln Phe Leu Pro Lys Thr Asp Ser Ile Leu Ser Asp Pro Thr 335 340 345Asp His Leu Gly Ile Glu Phe Met Glu Ser Glu Asp Val Tyr Gln 350 355 360Arg Gln 362 47 amino acids Amino Acid Linear NRG3 EGF-like domain/aminoacid seq. 1-47 4 His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala Tyr Cys LeuAsn 1 5 10 15 Asp Gly Glu Cys Phe Val Ile Glu Thr Leu Thr Gly Ser HisLys 20 25 30 His Cys Arg Cys Lys Glu Gly Tyr Gln Gly Val Arg Cys Asp Gln35 40 45 Phe Leu 47 2502 base pairs Nucleic Acid Single Linear HumanNRG3B1(hNRG3B1)/nucleic acid seq. 1-2502 5 TCACCGACCT AGTGGACTCCACTAGGTCGG TGGGCACGTA CTCCTTGACG 50 GAGCCCACCA CGATCCATTT GAGAAGCATGAGGCGCGGCC CCATGCCTCT 100 GCCGCGGCCC TCGGGGGGGC GAAGGTGAAN ACCGGCTCCTAGGATGAGTG 150 AAGGGGCGGC CGCTGCCTCG CCACCTGGTG CCGCTTCGGC AGCCGCCGCC200 TCGGCCGAGG AGGGCACCGC GGCGGCTGCG GCGGCGGCAG CGGCGGGCGG 250GGGCCCGGAC GGCGGCGGCG AAGGGGCGGC CGAGCCCCCC CGGGAGTTAC 300 GCTGTAGCGACTGCATCGTG TGGAACCGGC AGCAGACGTG GCTGTGCGTG 350 GTACCTCTGT TCATCGGCTTCATCGGCCTG GGGCTCAGCC TCATGCTTCT 400 CAAATGGATC GTGGTGGGCT CCGTCAAGGAGTACGTGCCC ACCGACCTAG 450 TGGACTCCAA GGGGATGGGC CAGGACCCCT TCTTCCTCTCCAAGCCCAGC 500 TCTTTCCCCA AGGCCATGGA GACCACCACC ACTACCACTT CCACCACGTC550 CCCCGCCACC CCCTCCGCCG GGGGTGCCGC CTCCTCCAGG ACGCCCAACC 600GGATTAGCAC TCGCCTGACC ACCATCACGC GGGCGCCCAC TCGCTTCCCC 650 GGGCACCGGGTGCCCATCCG GGCCAGCCCG CGCTCCACCA CAGCACGGAA 700 CACTGCGGCC CCTGCGACGGTCCCGTCCAC CACGGCCCCG TTCTTCAGTA 750 GCAGCACGCT GGGCTCCCGA CCCCCGGTGCCAGGAACTCC AAGTACCCAG 800 GCAATGCCCT CCTGGCCTAC TGCGGCATAC GCTACCTCCTCCTACCTTCA 850 CGATTCTACT CCCTCCTGGA CCCTGTCTCC CTTTCAGGAT GCTGCCTCCT900 CTTCTTCCTC TTCTTCCTCC TCCGCTACCA CCACCACACC AGAAACTAGC 950ACCAGCCCCA AATTTCATAC GACGACATAT TCCACAGAGC GATCCGAGCA 1000 CTTCAAACCCTGCCGAGACA AGGACCTTGC ATACTGTCTC AATGATGGCG 1050 AGTGCTTTGT GATCGAAACCCTGACCGGAT CCCATAAACA CTGTCGGTGC 1100 AAAGAAGGCT ACCAAGGAGT CCGTTGTGATCAATTTCTGC CGAAAACTGA 1150 TTCCATCTTA TCGGATCCAA CAGACCACTT GGGGATTGAATTCATGGAGA 1200 GTGAAGAAGT TTATCAAAGG CAGGTGCTGT CAATTTCATG TATCATCTTT1250 GGAATTGTCA TCGTGGGCAT GTTCTGTGCA GCATTCTACT TCAAAAGCAA 1300GAAACAAGCT AAACAAATCC AAGAGCAGCT GAAAGTGCCA CAAAATGGTA 1350 AAAGCTACAGTCTCAAAGCA TCCAGCACAA TGGCAAAGTC AGAGAACTTG 1400 GTGAAGAGCC ATGTCCAGCTGCAAAATTAT TCAAAGGTGG AAAGGCATCC 1450 TGTGACTGCA TTGGAGAAAA TGATGGAGTCAAGTTTTGTC GGCCCCCAGT 1500 CATTCCCTGA GGTCCCTTCT CCTGACAGAG GAAGCCAGTCTGTCAAACAC 1550 CACAGGAGTC TATCCTCTTG CTGCAGCCCA GGGCAAAGAA GTGGCATGCT1600 CCATAGGAAT GCCTTCAGAA GGACACCCCC GTCACCCCGA AGTAGGCTAG 1650GTGGAATTGT GGGACCAGCA TATCAGCAAC TCGAAGAATC AAGGATCCCA 1700 GACCAGGATACGATACCTTG CCAAGGGATA GAGGTCAGGA AGACTATATC 1750 CCACCTGCCT ATACAGCTGTGGTGTGTTGA AAGACCCCTG GACTTAAAGT 1800 ATTCATCCAG TGGTTTAAAA ACCCAACGAAATACATCAAT AAATATGCAA 1850 CTGCCTTCAA GAGAGACAAA CCCCTATTTT AATAGCTTGGAGCAAAAGGA 1900 CCTGGTGGGC TATTCATCCA CAAGGGCCAG TTCTGTGCCC ATCATCCCTT1950 CAGTGGGTTT AGAGGAAACC TGCCTGCAAA TGCCAGGGAT TTCTGAAGTC 2000AAAAGCATCA AATGGTGCAA AAACTCCTAT TCAGCTGACG TTGTCAATGT 2050 GAGTATTCCAGTCAGCGATT GTCTTATAGC AGAACAACAA GAAGTGAAAA 2100 TATTGCTAGA AACTGTCCAGGAGCAGATCC GAATTCTGAC TGATGCCAGA 2150 CGGTCAGAAG ACTACGAACT GGCCAGCGTAGAAACCGAGG ACAGTGCAAG 2200 CGAAAACACA GCCTTTCTCC CCCTGAGTCC CACAGCCAAATCAGAACGAG 2250 AGGCGCAATT TGTCTTAAGA AATGAAATAC AAAGAGACTC TGCATTGACC2300 AAGTGACTTG AGATGTAGGA ATCTGTGCAT TCTATGCTTT GCTCAACAGG 2350AAAGAGAGGA AATCAAATAC AAATTATTTA TATGCATTAA TTTAAGAGCA 2400 TCTACTTAGAAGAAACCAAA TAGTCTATCG CCCTCATATC ATAGTGTTTT 2450 TTAACAAAAT ATTTTTTTAAGGGAAAGAAA TGTTTCAGGA GGGATAAAGC 2500 TT 2502 720 amino acids Amino AcidLinear hNRG3B1 amino acid sequence 1-720 6 Met Ser Glu Gly Ala Ala AlaAla Ser Pro Pro Gly Ala Ala Ser 1 5 10 15 Ala Ala Ala Ala Ser Ala GluGlu Gly Thr Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Gly Gly Gly ProAsp Gly Gly Gly Glu Gly Ala 35 40 45 Ala Glu Pro Pro Arg Glu Leu Arg CysSer Asp Cys Ile Val Trp 50 55 60 Asn Arg Gln Gln Thr Trp Leu Cys Val ValPro Leu Phe Ile Gly 65 70 75 Phe Ile Gly Leu Gly Leu Ser Leu Met Leu LeuLys Trp Ile Val 80 85 90 Val Gly Ser Val Lys Glu Tyr Val Pro Thr Asp LeuVal Asp Ser 95 100 105 Lys Gly Met Gly Gln Asp Pro Phe Phe Leu Ser LysPro Ser Ser 110 115 120 Phe Pro Lys Ala Met Glu Thr Thr Thr Thr Thr ThrSer Thr Thr 125 130 135 Ser Pro Ala Thr Pro Ser Ala Gly Gly Ala Ala SerSer Arg Thr 140 145 150 Pro Asn Arg Ile Ser Thr Arg Leu Thr Thr Ile ThrArg Ala Pro 155 160 165 Thr Arg Phe Pro Gly His Arg Val Pro Ile Arg AlaSer Pro Arg 170 175 180 Ser Thr Thr Ala Arg Asn Thr Ala Ala Pro Ala ThrVal Pro Ser 185 190 195 Thr Thr Ala Pro Phe Phe Ser Ser Ser Thr Leu GlySer Arg Pro 200 205 210 Pro Val Pro Gly Thr Pro Ser Thr Gln Ala Met ProSer Trp Pro 215 220 225 Thr Ala Ala Tyr Ala Thr Ser Ser Tyr Leu His AspSer Thr Pro 230 235 240 Ser Trp Thr Leu Ser Pro Phe Gln Asp Ala Ala SerSer Ser Ser 245 250 255 Ser Ser Ser Ser Ser Ala Thr Thr Thr Thr Pro GluThr Ser Thr 260 265 270 Ser Pro Lys Phe His Thr Thr Thr Tyr Ser Thr GluArg Ser Glu 275 280 285 His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala TyrCys Leu Asn 290 295 300 Asp Gly Glu Cys Phe Val Ile Glu Thr Leu Thr GlySer His Lys 305 310 315 His Cys Arg Cys Lys Glu Gly Tyr Gln Gly Val ArgCys Asp Gln 320 325 330 Phe Leu Pro Lys Thr Asp Ser Ile Leu Ser Asp ProThr Asp His 335 340 345 Leu Gly Ile Glu Phe Met Glu Ser Glu Glu Val TyrGln Arg Gln 350 355 360 Val Leu Ser Ile Ser Cys Ile Ile Phe Gly Ile ValIle Val Gly 365 370 375 Met Phe Cys Ala Ala Phe Tyr Phe Lys Ser Lys LysGln Ala Lys 380 385 390 Gln Ile Gln Glu Gln Leu Lys Val Pro Gln Asn GlyLys Ser Tyr 395 400 405 Ser Leu Lys Ala Ser Ser Thr Met Ala Lys Ser GluAsn Leu Val 410 415 420 Lys Ser His Val Gln Leu Gln Asn Tyr Ser Lys ValGlu Arg His 425 430 435 Pro Val Thr Ala Leu Glu Lys Met Met Glu Ser SerPhe Val Gly 440 445 450 Pro Gln Ser Phe Pro Glu Val Pro Ser Pro Asp ArgGly Ser Gln 455 460 465 Ser Val Lys His His Arg Ser Leu Ser Ser Cys CysSer Pro Gly 470 475 480 Gln Arg Ser Gly Met Leu His Arg Asn Ala Phe ArgArg Thr Pro 485 490 495 Pro Ser Pro Arg Ser Arg Leu Gly Gly Ile Val GlyPro Ala Tyr 500 505 510 Gln Gln Leu Glu Glu Ser Arg Ile Pro Asp Gln AspThr Ile Pro 515 520 525 Cys Gln Gly Ile Glu Val Arg Lys Thr Ile Ser HisLeu Pro Ile 530 535 540 Gln Leu Trp Cys Val Glu Arg Pro Leu Asp Leu LysTyr Ser Ser 545 550 555 Ser Gly Leu Lys Thr Gln Arg Asn Thr Ser Ile AsnMet Gln Leu 560 565 570 Pro Ser Arg Glu Thr Asn Pro Tyr Phe Asn Ser LeuGlu Gln Lys 575 580 585 Asp Leu Val Gly Tyr Ser Ser Thr Arg Ala Ser SerVal Pro Ile 590 595 600 Ile Pro Ser Val Gly Leu Glu Glu Thr Cys Leu GlnMet Pro Gly 605 610 615 Ile Ser Glu Val Lys Ser Ile Lys Trp Cys Lys AsnSer Tyr Ser 620 625 630 Ala Asp Val Val Asn Val Ser Ile Pro Val Ser AspCys Leu Ile 635 640 645 Ala Glu Gln Gln Glu Val Lys Ile Leu Leu Glu ThrVal Gln Glu 650 655 660 Gln Ile Arg Ile Leu Thr Asp Ala Arg Arg Ser GluAsp Tyr Glu 665 670 675 Leu Ala Ser Val Glu Thr Glu Asp Ser Ala Ser GluAsn Thr Ala 680 685 690 Phe Leu Pro Leu Ser Pro Thr Ala Lys Ser Glu ArgGlu Ala Gln 695 700 705 Phe Val Leu Arg Asn Glu Ile Gln Arg Asp Ser AlaLeu Thr Lys 710 715 720 360 amino acids Amino Acid Linear hNRG3extracellular domain/Amino AcidSeq 1-360 7 Met Ser Glu Gly Ala Ala AlaAla Ser Pro Pro Gly Ala Ala Ser 1 5 10 15 Ala Ala Ala Ala Ser Ala GluGlu Gly Thr Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Ala Gly Gly Gly ProAsp Gly Gly Gly Glu Gly Ala 35 40 45 Ala Glu Pro Pro Arg Glu Leu Arg CysSer Asp Cys Ile Val Trp 50 55 60 Asn Arg Gln Gln Thr Trp Leu Cys Val ValPro Leu Phe Ile Gly 65 70 75 Phe Ile Gly Leu Gly Leu Ser Leu Met Leu LeuLys Trp Ile Val 80 85 90 Val Gly Ser Val Lys Glu Tyr Val Pro Thr Asp LeuVal Asp Ser 95 100 105 Lys Gly Met Gly Gln Asp Pro Phe Phe Leu Ser LysPro Ser Ser 110 115 120 Phe Pro Lys Ala Met Glu Thr Thr Thr Thr Thr ThrSer Thr Thr 125 130 135 Ser Pro Ala Thr Pro Ser Ala Gly Gly Ala Ala SerSer Arg Thr 140 145 150 Pro Asn Arg Ile Ser Thr Arg Leu Thr Thr Ile ThrArg Ala Pro 155 160 165 Thr Arg Phe Pro Gly His Arg Val Pro Ile Arg AlaSer Pro Arg 170 175 180 Ser Thr Thr Ala Arg Asn Thr Ala Ala Pro Ala ThrVal Pro Ser 185 190 195 Thr Thr Ala Pro Phe Phe Ser Ser Ser Thr Leu GlySer Arg Pro 200 205 210 Pro Val Pro Gly Thr Pro Ser Thr Gln Ala Met ProSer Trp Pro 215 220 225 Thr Ala Ala Tyr Ala Thr Ser Ser Tyr Leu His AspSer Thr Pro 230 235 240 Ser Trp Thr Leu Ser Pro Phe Gln Asp Ala Ala SerSer Ser Ser 245 250 255 Ser Ser Ser Ser Ser Ala Thr Thr Thr Thr Pro GluThr Ser Thr 260 265 270 Ser Pro Lys Phe His Thr Thr Thr Tyr Ser Thr GluArg Ser Glu 275 280 285 His Phe Lys Pro Cys Arg Asp Lys Asp Leu Ala TyrCys Leu Asn 290 295 300 Asp Gly Glu Cys Phe Val Ile Glu Thr Leu Thr GlySer His Lys 305 310 315 His Cys Arg Cys Lys Glu Gly Tyr Gln Gly Val ArgCys Asp Gln 320 325 330 Phe Leu Pro Lys Thr Asp Ser Ile Leu Ser Asp ProThr Asp His 335 340 345 Leu Gly Ile Glu Phe Met Glu Ser Glu Glu Val TyrGln Arg Gln 350 355 360 47 amino acids Amino Acid Linear NRG3 EGF-likedomain/amino acid seq. 1-47 8 His Phe Lys Pro Cys Arg Asp Lys Asp LeuAla Tyr Cys Leu Asn 1 5 10 15 Asp Gly Glu Cys Phe Val Ile Glu Thr LeuThr Gly Ser His Lys 20 25 30 His Cys Arg Cys Lys Glu Gly Tyr Gln Gly ValArg Cys Asp Gln 35 40 45 Phe Leu 47 48 amino acids Amino Acid LinearcARIA.egf 1-48 9 His Leu Thr Lys Cys Asp Ile Lys Gln Lys Ala Phe Cys ValAsn 1 5 10 15 Gly Gly Glu Cys Tyr Met Val Lys Asp Leu Pro Asn Pro ProArg 20 25 30 Tyr Leu Cys Arg Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln35 40 45 Asn Tyr Val 48 45 amino acids Amino Acid Linear hAR.egf 1-45 10Lys Lys Asn Pro Cys Asn Ala Glu Phe Gln Asn Phe Cys Ile His 1 5 10 15Gly Glu Cys Lys Tyr Ile Glu His Leu Glu Ala Val Thr Cys Lys 20 25 30 CysGln Gln Glu Tyr Phe Gly Glu Arg Cys Gly Glu Lys Ser Met 35 40 45 45amino acids Amino Acid Linear hBTC.efg 1-45 11 His Phe Ser Arg Cys ProLys Gln Tyr Lys His Tyr Cys Ile Lys 1 5 10 15 Gly Arg Cys Arg Phe ValVal Ala Glu Gln Thr Pro Ser Cys Val 20 25 30 Cys Asp Glu Gly Tyr Ile GlyAla Arg Cys Glu Arg Val Asp Leu 35 40 45 46 amino acids Amino AcidLinear hEGF.egf 1-46 12 Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly TyrCys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp LysTyr Ala Cys 20 25 30 Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln TyrArg Asp 35 40 45 Leu 46 45 amino acids Amino Acid Linear hHB-EGF.egf1-45 13 Lys Arg Asp Pro Cys Leu Arg Lys Tyr Lys Asp Phe Cys Ile His 1 510 15 Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg Ala Pro Ser Cys Ile 20 2530 Cys His Pro Gly Tyr His Gly Glu Arg Cys His Gly Leu Ser Leu 35 40 4549 amino acids Amino Acid Linear hHRGalpha.egf 1-49 14 His Leu Val LysCys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn 1 5 10 15 Gly Gly Glu CysPhe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 20 25 30 Tyr Leu Cys Lys CysGln Pro Gly Phe Thr Gly Ala Arg Cys Thr 35 40 45 Glu Asn Tyr Pro 49 48amino acids Amino Acid Linear hHRGbeta.egf 1-48 15 His Leu Val Lys CysAla Glu Lys Glu Lys Thr Phe Cys Val Asn 1 5 10 15 Gly Gly Glu Cys PheMet Val Lys Asp Leu Ser Asn Pro Ser Arg 20 25 30 Tyr Leu Cys Lys Cys ProAsn Glu Phe Thr Gly Asp Arg Cys Gln 35 40 45 Asn Tyr Val 48 45 aminoacids Amino Acid Linear hTGFalpha.egf 1-45 16 His Phe Asn Asp Cys ProAsp Ser His Thr Gln Phe Cys Phe His 1 5 10 15 Gly Thr Cys Arg Phe LeuVal Gln Glu Asp Lys Pro Ala Cys Val 20 25 30 Cys His Ser Gly Tyr Val GlyAla Arg Cys Glu His Ala Asp Leu 35 40 45 45 amino acids Amino AcidLinear mEPR.egf 1-45 17 Gln Ile Thr Lys Cys Ser Ser Asp Met Asp Gly TyrCys Leu His 1 5 10 15 Gly Gln Cys Ile Tyr Leu Val Asp Met Arg Glu LysPhe Cys Arg 20 25 30 Cys Glu Val Gly Tyr Thr Gly Leu Arg Cys Glu His PhePhe Leu 35 40 45 50 amino acids Amino Acid Linear Oligonucleotide probe1-50 18 Thr Gly Gly Thr Ala Ala Ala Ala Gly Cys Thr Ala Cys Ala Gly 1 510 15 Thr Cys Thr Cys Ala Ala Ala Gly Cys Ala Thr Cys Cys Ala Gly 20 2530 Cys Ala Cys Ala Ala Thr Gly Gly Cys Ala Ala Ala Gly Thr Cys 35 40 45Ala Gly Ala Gly Ala 50 8 amino acids Amino Acid Linear hNRG3B1transmembrane proximal 1 1-8 19 Asn Asp Gly Glu Cys Phe Val Ile 1 5 8 9amino acids Amino Acid Linear hNRG3B1 transmembrane proximal 2 1-9 20Glu Phe Met Glu Ser Glu Glu Val Tyr 1 5 9 466 base pairs Nucleic AcidSingle Linear EST Genbank entry H23651 1-466 21 AATTTCTGCC GAAAACTGATTCCATCTTAT CGGATCCAAC AGACCACTTG 50 GGGATTGAAT TCATGGAGAG TGAAGAAGTTTATCAAAGGC AGGTGCTGTC 100 AATTTCATGT ATCATCTTTG GAATTGTCAT CGTGGGCATGTTCTGTGCAG 150 CATTCTACTT CAAAAGCAAG AAACAAGCTA AACAAATCCA AGAGCAGCTG200 AAAGTGCCAC AAAATGGTAA AAGCTACAGT CTCAAAGCAT CCAGCACAAT 250GGCAAAGTCA GAGAACTTGG TGAAGAGCCA TGTCCAGCTG CAAAATAAAA 300 TGTCAGGCTTCTGAGCCCAA GCTAAGCCAT CATATCCCCT GTNGACCTGC 350 ACGTGCACAT CCNGATGGCCCGTTTCCTGC CTTTTNTGAT GACATTTNCA 400 CCACAAATGN AGTGAAAATG GGNCTTTTCNTGCCTTAACT GGTTGACNTT 450 TTTNCCCCAA AAGGAG 466 2091 base pairs NucleicAcid Single Linear 22 ATGAGTGAAG GGGCGGCCGC TGCCTCGCCA CCTGGTGCCGCTTCGGCAGC 50 CGCCGCCTCG GCCGAGGAGG GCACCGCGGC GGCTGCGGCG GCGGCAGCGG 100CGGGCGGGGG CCCGGACGGC GGCGGCGAAG GGGCGGCCGA GCCCCCCCGG 150 GAGTTACGCTGTAGCGACTG CATCGTGTGG AACCGGCAGC AGACGTGGCT 200 GTGCGTGGTA CCTCTGTTCATCGGCTTCAT CGGCCTGGGG CTCAGCCTCA 250 TGCTTCTCAA ATGGATCGTG GTGGGCTCCGTCAAGGAGTA CGTGCCCACC 300 GACCTAGTGG ACTCCAAGGG GATGGGCCAG GACCCCTTCTTCCTCTCCAA 350 GCCCAGCTCT TTCCCCAAGG CCATGGAGAC CACCACCACT ACCACTTCCA400 CCACGTCCCC CGCCACCCCC TCCGCCGGGG GTGCCGCCTC CTCCAGGACG 450CCCAACCGGA TTAGCACTCG CCTGACCACC ATCACGCGGG CGCCCACTCG 500 CTTCCCCGGGCACCGGGTGC CCATCCGGGC CAGCCCGCGC TCCACCACAG 550 CACGGAACAC TGCGGCCCCTGCGACGGTCC CGTCCACCAC GGCCCCGTTC 600 TTCAGTAGCA GCACGCTGGG CTCCCGACCCCCGGTGCCAG GAACTCCAAG 650 TACCCAGGCA ATGCCCTCCT GGCCTACTGC GGCATACGCTACCTCCTCCT 700 ACCTTCACGA TTCTACTCCC TCCTGGACCC TGTCTCCCTT TCAGGATGCT750 GCCTCCTCTT CTTCCTCTTC TTCCTCCTCC GCTACCACCA CCACACCAGA 800AACTAGCACC AGCCCCAAAT TTCATACGAC GACATATTCC ACAGAGCGAT 850 CCGAGCACTTCAAACCCTGC CGAGACAAGG ACCTTGCATA CTGTCTCAAT 900 GATGGCGAGT GCTTTGTGATCGAAACCCTG ACCGGATCCC ATAAACACTG 950 TCGGTGCAAA GAAGGCTACC AAGGAGTCCGTTGTGATCAA TTTCTGCCGA 1000 AAACTGATTC CATCTTATCG GATCCAACAG ACCACTTGGGGATTGAATTC 1050 ATGGAGAGTG AAGAAGTTTA TCAAAGGCAG GTGCTGTCAA TTTCATGTAT1100 CATCTTTGGA ATTGTCATCG TGGGCATGTT CTGTGCAGCA TTCTACTTCA 1150AAAGCAAGAA ACAAGCTAAA CAAATCCAAG AGCAGCTGAA AGTGCCACAA 1200 AATGGTAAAAGCTACAGTCT CAAAGCATCC AGCACAATGG CAAAGTCAGA 1250 GAACTTGGTG AAGAGCCATGTCCAGCTGCA AAATTATTCA AAGGTGGAAA 1300 GGCATCCTGT GACTGCATTG GAGAAAATGATGGAGTCAAG TTTTGTCGGC 1350 CCCCAGTCAT TCCCTGAGGT CCCTTCTCCT GACAGAGGAAGCCAGTCTGT 1400 CAAACACCAC AGGAGTCTAT CCTCTTGCTG CAGCCCAGGG CAAAGAAGTG1450 GCATGCTCCA TAGGAATGCC TTCAGAAGGA CACCCCCGTC ACCCCGAAGT 1500AGGCTAGGTG GAATTGTGGG ACCAGCATAT CAGCAACTCG AAGAATCAAG 1550 GATCCCAGACCAGGATACGA TACCTTGCCA AGGGTATTCA TCCAGTGGTT 1600 TAAAAACCCA ACGAAATACATCAATAAATA TGCAACTGCC TTCAAGAGAG 1650 ACAAACCCCT ATTTTAATAG CTTGGAGCAAAAGGACCTGG TGGGCTATTC 1700 ATCCACAAGG GCCAGTTCTG TGCCCATCAT CCCTTCAGTGGGTTTAGAGG 1750 AAACCTGCCT GCAAATGCCA GGGATTTCTG AAGTCAAAAG CATCAAATGG1800 TGCAAAAACT CCTATTCAGC TGACGTTGTC AATGTGAGTA TTCCAGTCAG 1850CGATTGTCTT ATAGCAGAAC AACAAGAAGT GAAAATATTG CTAGAAACTG 1900 TCCAGGAGCAGATCCGAATT CTGACTGATG CCAGACGGTC AGAAGACTAC 1950 GAACTGGCCA GCGTAGAAACCGAGGACAGT GCAAGCGAAA ACACAGCCTT 2000 TCTCCCCCTG AGTCCCACAG CCAAATCAGAACGAGAGGCG CAATTTGTCT 2050 TAAGAAATGA AATACAAAGA GACTCTGCAT TGACCAAGTG A2091 696 amino acids Amino Acid Linear Human NRG3B2 1-696 23 Met Ser GluGly Ala Ala Ala Ala Ser Pro Pro Gly Ala Ala Ser 1 5 10 15 Ala Ala AlaAla Ser Ala Glu Glu Gly Thr Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala AlaGly Gly Gly Pro Asp Gly Gly Gly Glu Gly Ala 35 40 45 Ala Glu Pro Pro ArgGlu Leu Arg Cys Ser Asp Cys Ile Val Trp 50 55 60 Asn Arg Gln Gln Thr TrpLeu Cys Val Val Pro Leu Phe Ile Gly 65 70 75 Phe Ile Gly Leu Gly Leu SerLeu Met Leu Leu Lys Trp Ile Val 80 85 90 Val Gly Ser Val Lys Glu Tyr ValPro Thr Asp Leu Val Asp Ser 95 100 105 Lys Gly Met Gly Gln Asp Pro PhePhe Leu Ser Lys Pro Ser Ser 110 115 120 Phe Pro Lys Ala Met Glu Thr ThrThr Thr Thr Thr Ser Thr Thr 125 130 135 Ser Pro Ala Thr Pro Ser Ala GlyGly Ala Ala Ser Ser Arg Thr 140 145 150 Pro Asn Arg Ile Ser Thr Arg LeuThr Thr Ile Thr Arg Ala Pro 155 160 165 Thr Arg Phe Pro Gly His Arg ValPro Ile Arg Ala Ser Pro Arg 170 175 180 Ser Thr Thr Ala Arg Asn Thr AlaAla Pro Ala Thr Val Pro Ser 185 190 195 Thr Thr Ala Pro Phe Phe Ser SerSer Thr Leu Gly Ser Arg Pro 200 205 210 Pro Val Pro Gly Thr Pro Ser ThrGln Ala Met Pro Ser Trp Pro 215 220 225 Thr Ala Ala Tyr Ala Thr Ser SerTyr Leu His Asp Ser Thr Pro 230 235 240 Ser Trp Thr Leu Ser Pro Phe GlnAsp Ala Ala Ser Ser Ser Ser 245 250 255 Ser Ser Ser Ser Ser Ala Thr ThrThr Thr Pro Glu Thr Ser Thr 260 265 270 Ser Pro Lys Phe His Thr Thr ThrTyr Ser Thr Glu Arg Ser Glu 275 280 285 His Phe Lys Pro Cys Arg Asp LysAsp Leu Ala Tyr Cys Leu Asn 290 295 300 Asp Gly Glu Cys Phe Val Ile GluThr Leu Thr Gly Ser His Lys 305 310 315 His Cys Arg Cys Lys Glu Gly TyrGln Gly Val Arg Cys Asp Gln 320 325 330 Phe Leu Pro Lys Thr Asp Ser IleLeu Ser Asp Pro Thr Asp His 335 340 345 Leu Gly Ile Glu Phe Met Glu SerGlu Glu Val Tyr Gln Arg Gln 350 355 360 Val Leu Ser Ile Ser Cys Ile IlePhe Gly Ile Val Ile Val Gly 365 370 375 Met Phe Cys Ala Ala Phe Tyr PheLys Ser Lys Lys Gln Ala Lys 380 385 390 Gln Ile Gln Glu Gln Leu Lys ValPro Gln Asn Gly Lys Ser Tyr 395 400 405 Ser Leu Lys Ala Ser Ser Thr MetAla Lys Ser Glu Asn Leu Val 410 415 420 Lys Ser His Val Gln Leu Gln AsnTyr Ser Lys Val Glu Arg His 425 430 435 Pro Val Thr Ala Leu Glu Lys MetMet Glu Ser Ser Phe Val Gly 440 445 450 Pro Gln Ser Phe Pro Glu Val ProSer Pro Asp Arg Gly Ser Gln 455 460 465 Ser Val Lys His His Arg Ser LeuSer Ser Cys Cys Ser Pro Gly 470 475 480 Gln Arg Ser Gly Met Leu His ArgAsn Ala Phe Arg Arg Thr Pro 485 490 495 Pro Ser Pro Arg Ser Arg Leu GlyGly Ile Val Gly Pro Ala Tyr 500 505 510 Gln Gln Leu Glu Glu Ser Arg IlePro Asp Gln Asp Thr Ile Pro 515 520 525 Cys Gln Gly Tyr Ser Ser Ser GlyLeu Lys Thr Gln Arg Asn Thr 530 535 540 Ser Ile Asn Met Gln Leu Pro SerArg Glu Thr Asn Pro Tyr Phe 545 550 555 Asn Ser Leu Glu Gln Lys Asp LeuVal Gly Tyr Ser Ser Thr Arg 560 565 570 Ala Ser Ser Val Pro Ile Ile ProSer Val Gly Leu Glu Glu Thr 575 580 585 Cys Leu Gln Met Pro Gly Ile SerGlu Val Lys Ser Ile Lys Trp 590 595 600 Cys Lys Asn Ser Tyr Ser Ala AspVal Val Asn Val Ser Ile Pro 605 610 615 Val Ser Asp Cys Leu Ile Ala GluGln Gln Glu Val Lys Ile Leu 620 625 630 Leu Glu Thr Val Gln Glu Gln IleArg Ile Leu Thr Asp Ala Arg 635 640 645 Arg Ser Glu Asp Tyr Glu Leu AlaSer Val Glu Thr Glu Asp Ser 650 655 660 Ala Ser Glu Asn Thr Ala Phe LeuPro Leu Ser Pro Thr Ala Lys 665 670 675 Ser Glu Arg Glu Ala Gln Phe ValLeu Arg Asn Glu Ile Gln Arg 680 685 690 Asp Ser Ala Leu Thr Lys 695 696

What is claimed is:
 1. A polypeptide comprising an amino acid sequenceencoding an EGF-like domain, wherein the amino acid sequence has thebinding characteristics of NRG3.
 2. The polypeptide of claim 1 whereinthe binding characteristics of NRG3 comprise (a) binding to ErbB4receptor but not to ErbB2 receptor or ErbB3 receptor underexperimentally comparable conditions; and (b) activation of ErbB4receptor tyrosine phosphorylation.
 3. The polypeptide of claim 1 whereinthe amino acid sequence has at least 75% amino acid sequence homology tothe amino acid sequence SEQ ID NO:
 4. 4. The polypeptide of claim 1,wherein the polypeptide binds to the ErbB4 receptor and stimulatestyrosine phosphorylation of the ErbB4 receptor.
 5. A polypeptide thatbinds ErbB4 receptor, which polypeptide is selected from the groupconsisting of (a) a polypeptide comprising an amino acid sequence havingat least 75% sequence homology to the extracellular domain NRG3 (SEQ IDNO: 3 or 7). (b) a polypeptide comprising an amino acid sequence havingat least 75% sequence homology to SEQ ID NO: 2 or SEQ ID NO: 6; (c) afurther mammalian homologue of polypeptide (a) or (b): (d) a solubleform of any of the polypeptides (a)- (c) having a transmembrane domainthat cannot anchor the polypeptide in a cell membrane; and (e) aderivative of any of the polypeptides (a)- (d) having the bindingcharacteristics of NRG3.
 6. The polypeptide of claim 1 encoded by a NRG3nucleic acid open reading frame sequence in ATCC deposit 209156(pLXSN.mNRG3).
 7. The polypeptide of claim 1 encoded by a NRG3 nucleicacid open reading frame sequence in ATCC deposit 209157(pRK5.tk.neo.hNRG3B1).
 8. The polypeptide of claim 1 encoded by a NRG3nucleic acid open reading frame sequence in ATCC deposit 209297(pRK5.tk.neo.hNRG3B2).
 9. The polypeptide of claim 1 which is devoid ofa cytoplasmic domain, or devoid of a transmembrane domain that cananchor the polypeptide in a cell membrane, or both.
 10. The polypeptideof claim 1 unaccompanied by native glycosylation.
 11. The polypeptide ofclaim 1 which has a variant glycosylation.
 12. An antagonist of thepolypeptide of claim
 1. 13. An agonist of the polypeptide of claim 1.14. An isolated nucleic acid molecule encoding the polypeptide ofclaim
 1. 15. The nucleic acid molecule of claim 14 further encoding theextracellular domain of a mammalian NRG3.
 16. The nucleic acid moleculeof claim 15, wherein the encoded extracellular domain has at least 75%amino acid sequence identity to the amino acid sequence of SEQ ID NO: 3or SEQ ID NO:
 7. 17. The nucleic acid molecule of claim 14 wherein theencoded amino acid sequence is devoid of a cytoplasmic domain or atransmembrane domain that can anchor the polypeptide in a cell membrane,or both.
 18. An expression vector comprising the nucleic acid moleculeof claim 14 operably linked to control sequences recognized by a hostcell transformed with the vector.
 19. An expression vector according toclaim 18 obtainable as ATCC 209156 (pLXSN.mNRG3).
 20. An expressionvector according to claim 18 obtainable as ATCC 209157(pRK5.tk.neo.hNRG3B1).
 21. An expression vector according to claim 18obtainable as ATCC 209297 (pRK5.tk.neo.hNRG3B2).
 22. A host cellcomprising the vector of claim
 18. 23. The host cell of claim 22 whichis a mammalian cell.
 24. The host cell of claim 23 which is a Chinesehamster ovary cell line.
 25. A method for producing the amino acidsequence encoding an EGF-like domain that binds ErbB4 receptor, themethod comprising: a) culturing a cell comprising the nucleic acid ofclaim 14; and b) recovering the polypeptide from the cell culture. 26.The method of claim 25 wherein the polypeptide is secreted into theculture medium and recovered from the culture medium.
 27. An antibodythat specifically binds to the polypeptide of claim
 1. 28. A hybridomacell line producing the antibody of claim
 27. 29. An immunoadhesincomprising the polypeptide of claim 1 fused to an immunoglobulinsequence.
 30. The immunoadhesin of claim 29, further comprising theEGF-like domain of SEQ ID NO:
 4. 31. The immunoadhesin of claim 29wherein the immunoglobulin sequence is an immunoglobulin heavy chainconstant domain sequence.
 32. The immunoadhesin of claim 31 wherein theimmunoglobulin sequence is a constant domain sequence of an IgG-1, IgG-2or IgG-3.
 33. A method of detecting an NRG3 in a sample, the methodcomprising: a) contacting the antibody of claim 27 with the sample; b)detecting binding of the antibody to a polypeptide in the sample,wherein the polypeptide is an NRG3.
 34. A method of detecting ErbB4receptor in a sample, the method comprising: a) contacting thepolypeptide of claim 1 with the sample; and b) detecting binding of theamino acid sequence to a protein in the sample.
 35. The method of claim34 wherein the sample comprises a cell expressing ErB4 receptor on itssurface.
 36. The method of claim 35 wherein the sample is a mammaliantissue sample.
 37. A method of administering a NRG3 polypeptide to amammal experiencing a disorder treatable with NRG3, wherein the methodcomprises introducing into the mammal a cell comprising the nucleic acidof claim 14, and wherein the NRG3 polypeptide is secreted by the cell.38. The method of claim 37 wherein the cell is contained within a porousmatrix and the matrix is administered to the mammal.